KR20210013571A - Vaccine composition - Google Patents

Abstract

The present invention relates to vaccine compositions, in particular to vaccine compositions having a large antigenic component, for example greater than 50 kDa, or multimer, ie comprising subunits. These antigenic components are of particular importance because they can represent antigenic components from pathogens that are currently not vaccinated. The present invention relates to a composition comprising particles representing an antigenic component, wherein the composition comprises an antigenic component comprising a first peptide tag and a moiety comprising a second peptide tag, wherein the antigenic component and moiety T is linked via isopeptide bonds between the first and second peptide tags, the antigenic component exceeding 50 kDa, or alternatively multimer.

Description

Vaccine composition

Vaccines are a safe and effective way to fight and eradicate infectious diseases. Vaccine development has been very successful, but there is a list of challenges for the remaining diseases for which the current vaccine does not exist, including many important pathogens representing threatening immune disorders. It is generally believed that an effective vaccine should last for a sufficient time to travel to the lymph nodes and generate an immune response.

Vaccine development aims to generate the necessary protective immune response while avoiding the risks inherent by using attenuated strains, the smaller antigenic components of these pathogens from the use of attenuated or killed pathogens. Proceeded to use. Emphasis on attempts to express the immunogenic portion of pathogen components (e.g. components required for pathogens to infect cells) that were simply restricted to short/small peptides and proteins due to technical issues with the expression of large or multi-component antigens. Put. However, a potential problem with the use of very short or small peptides is the risk of antigenic variable pathogens, which avoids the immune response induced by vaccination through changes in certain parts of the antigen. .

There will be immunological benefits associated with the expression of large/multicomponent antigens, including the ability to generate antibodies against multiple neutralizing epitopes of one pathogen. However, the expression of one or more large antigens capable of complexing remains a great challenge in such a way that the relevant antigenic epitope is maintained and presented for the generation of an effective antibody response. Therefore, there is still a need for improved methods for expressing large antigens and/or multicomponent antigens in a manner that can elicit a clinically significant immune response.

Previous recombinant vaccines designed to elicit an immune response against multiple antigenic components rely on each component being expressed and individually packaged in separate particles, e.g. anti-HPV vaccine Cervarix and In the case of Gardasil, the recombinant major capsid L1 protein of a specific HPV strain is expressed individually and assembled into virus-like particles (VLPs), and then various types of VLPs are combined into a vaccine formulation. As an alternative, multiple short epitopes are selected and combined into a single recombinant vaccine (e.g., Multimeric-001 influenza vaccine), but due to their nature, these epitopes can reduce the manufacturing complexity associated with three-dimensional structure or refolding. Since it is a short linear peptide chosen to avoid, no attempt has been made to represent a natural pathogen as presented to the immune system in active infection.

As an example, β-herpes human cytomegalovirus (human Cytomegalovirus: HCMV, also known as human herpesvirus-5 (HHV-5)) is a major viral cause of neonatal developmental disorders. These ubiquitous viruses have infected more than 60% of the general population, and the initial infection is usually mild or asymptomatic. After infection, the virus remains latent in the body, but can cause serious illness in immunocompromised patients (ie, HIV patients, transplant patients and patients receiving chemotherapy) or in the elderly. HCMV is the leading cause of infection in birth defects in developed countries. Up to 4 out of 200 infants are born with HCMV from congenital infections, with up to 10% of these having long-term consequences. HCMV infection is also associated with hypertension and atherosclerosis in adults (Cheng et al. (May 2009). Fruh K, ed. "Cytomegalovirus infection causes an increase of arterial blood pressure". PLoS Pathog. 5 ( 5): e1000427). Therefore, HCMV is a public health priority. However, despite intensive efforts, no successful HCMV vaccine has been developed to date.

Respiratory syncytial virus (RSV) is another ubiquitous virus that has little impact on the health of healthy adults and older children infected with this virus. However, it is the second-largest cause of death in infants under the age of one in the world after malaria. The virus kills about 160,000 people each year worldwide. This virus causes serious respiratory infections, and complications include pneumonia and bronchiolitis. The high-risk group includes infants under 1 year of age and immunocompromised patients, the elderly, and people with heart and lung disease. In other words, despite years of active research and development, there are currently no licensed RSV vaccines.

In the case of diseases such as diseases caused by RSV and HCMV for which no currently available vaccines are available, current approaches to vaccine manufacturing in general do not yield the desired efficacy, so alternative types of vaccines are used to deal with diseases that have these catastrophic consequences. There are many unmet needs in providing.

Recently several gene-encoded systems have been described that allow the formation of spontaneous or auxiliary amide bonds. For example, SpyTag is a peptide engineered to form spontaneous and irreversible isopeptide bonds to its protein partner SpyCatcher when the two components are mixed. The positions of the SpyTag and SpyCatcher components within the protein chain can be designed to be in a variety of positions and are reactive under a wide range of pH, buffer and temperature conditions. The SpyTag/SpyCatcher pair and its variants and derivatives have been used for vaccine development, but to date have been used only for the presentation of simple antigens. Other genetic coding systems that allow spontaneous amide bond formation include SnoopTag/SnoopTagJr and SnoopCatcher; RrgATag/RrgATag2/DogTag and RrgACatcher, IsopepTag/IsopepTag-N and Pilin-C or Pilin-N, PsCsTag and PsCsCatcher; And SnoopTagJr and DogTag (mediated by SnoopLigase), and variants of all of these systems.

The present inventors have demonstrated that using a gene-coding system that enables amide bond formation, the use of large/multi-component antigens in vaccine compositions is possible, which can improve the response to large/multi-component antigens. This is a surprising result.

In a first aspect of the invention, a composition is provided comprising particles representing a protein component, the composition comprising:

i) a protein component comprising a first peptide tag, and

ii) a moiety comprising a second peptide tag,

The protein component and moiety are linked via isopeptide bonds between the first and second peptide tags, and the protein component is greater than 50 kDa.

In another aspect of the invention, there is provided a composition comprising particles representing a protein component, the composition comprising:

i) a protein component comprising a first peptide tag, and

ii) a moiety comprising a second peptide tag,

The protein component and moiety are linked through an isopeptide bond between the first and second peptide tags, and the protein component is multimeric.

The protein component may have any function, such as it may be an enzyme or may have enzymatic properties. The protein component may be a full-length protein, or it may be a portion, segment, domain or cut of a full-length protein. The protein component may be an antigen or an immunogen. The protein component may also be referred to as an antigenic component.

In another aspect of the invention, there is provided a composition comprising particles exhibiting an antigenic component, the composition comprising:

iii) an antigenic component comprising a first peptide tag, and

iv) a moiety comprising a second peptide tag,

The antigenic component and moiety are linked via isopeptide bonds between the first and second peptide tags, and the antigenic component is greater than approximately 50 kDa.

In some embodiments of any aspect of the invention, the protein component or antigenic component is 60 kDa, 70 kDa, 80 kDa, 90 kDa, 100 kDa, 110 kDa, 120 kDa, 130 kDa, 140 kDa, 150 kDa, 160 kDa, 170 kDa, 180 kDa, 190 kDa or more, such as more than 200 kDa, more than 300 kDa or more than 400 kDa.

Multimers may contain any number of subunits, which may or may not be covalently linked in a protein or antigenic component. The multimer may comprise 2-20 subunits, alternatively 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more subunits. As an alternative, the multimer may be a dimer, a trimer, a tetramer, a pentamer, a hexamer, a heptomer, an octal, a 9mer, or a 10mer. The multimer may be derived from any suitable pathogen, but is preferably a viral multimer.

Non-limiting examples of large protein components above 50 kDa or described above include pentameric complexes (PC) and gB glycoproteins derived from human cytomegalovirus (HCMV), G and F glycoproteins derived from RSV, derived from influenza A virus. the hemagglutinin (HA) and neuraminidase (NA), plasminogen modium eight Shifa Room (Plasmodium falciparum ) Pfs230, P. falciparum CSP, human HER2 receptor, PCSK9, VAR2CSA, P. falciparum RIPR, Varicella zoster virus (VZV) glycoprotein E, rabies virus glycoprotein And EBV (Epstein-Barr virus) gH/gL complex.

In some embodiments of any aspect of the invention, the protein component or antigenic component may be a monomer or multimer, such as a dimer, trimer, tetramer or pentamer. In some embodiments of any aspect of the invention, the protein component or antigenic component may be a protein or peptide complex.

Non-limiting examples of multimeric antigenic components include pentameric complexes (PC) and gB trimers from human cytomegalovirus (HCMV), G and F glycoproteins from RSV, hemagglutinin from influenza A virus ( HA) and neuraminidase (NA) antigens, some of which are described herein. Other examples include components of disease agents such as viruses, bacteria, fungal pathogens, parasites or other disease vectors. Suitable multimeric antigenic components include, for example, those derived from viruses such as influenza (such as influenza hemagglutinin (HA) (such as Flu trimer)), respiratory syncytial virus (RSV), and the like.

The protein component may be attached to the first peptide tag through gene fusion, and may be recombinantly expressed in an appropriate cell. For components that contain post-translational modifications such as glycosylation, it may be desirable to express the recombinant protein in eukaryotic or mammalian cell lines.

In one embodiment, the "moiety" is a component in which a protein component or an antigenic component can be marked as available, for example to the immune system. In one embodiment, the moiety is multimerized to form the particle. Suitably, such moieties may be viruses, bacteria, multimerization scaffolds for vaccination or protein components that are multimerized to form VLPs (viral-like particles). Suitably, the moiety may be a component such as bacteriophage, tobacco mosaic virus particles, adeno-associated virus-like particles (AAVLP), E. coli. In one embodiment, the moiety is itself a component such as a virus, bacteria, etc., so that the multimerization (eg, self-assembly) of the moiety forms a particle for representing a protein component or an antigenic component. In one embodiment, the moiety may be a viral structural protein, for example a viral envelope or capsid protein or a surface antigen. Examples of structural proteins include matrix M1 protein and viral envelope M2 protein derived from influenza virus, HBsAg derived from hepatitis B virus, E. coli bacteriophage AP205 virus coat protein (CP3), and hemagglutinin derived from various viruses, including Mumps. -Includes neuraminidase and the like. Suitable viral structural proteins will be known to those of skill in the art. In another embodiment, the moiety may be a computationally derived particle such as MI3, or a protein or peptide such as a multimerization domain such as IMX313 forming nanoparticles. In a further embodiment, the moiety may be a synthetic nanoparticle or a synthetic VLP, such as gold, lipopeptide or poly(lactic-co-glycolic acid) (PLGA) nanoparticles. Other suitable moieties may include liposomes or outer membrane vesicles. Suitably, the moiety comprising the second peptide tag is a moiety to which the second peptide tag is attached.

It may be desirable to use structural surface antigens derived from viruses. Thus, a second peptide tag is attached to the structural surface antigen, allowing the formation of a virus-like particle (VLP) to which a second peptide tag is attached or exhibits a second peptide tag. VLPs are non-infectious self-assembled nanoparticles, and their repetitive molecule-defined structures are particularly attractive for manipulating multivalency for vaccination. VLPs include hepatitis B virus (including hepatitis B small surface antigen (HBsAg)), Parvoviride (e.g. adeno-associated virus), Retroviridae (e.g. HIV), flaviviride (Flaviviridae) (eg hepatitis C virus) and bacteriophage (eg Qβ, AP205). Either of these may be suitable for use as a moiety in the present invention.

The second peptide tag may be attached to the moiety through gene fusion. Such gene fusions are not limited to simply the ends, but can be at any suitable point in the sequence. One of skill in the art will understand that the fusion protein can be expressed recombinantly in appropriate cells.

The second peptide tag may alternatively be marked or attached to the moiety by chemical conjugation. This will require the presence of reactive amine groups, for example to allow conjugation to occur.

Thus, in one embodiment, the moiety is a surface antigen of hepatitis B virus (HBsAg). Suitably, the HBsAg has the amino acid sequence (or functional equivalent thereof) set forth in SEQ ID NO: 41 as described herein.

In one embodiment of any aspect of the invention, the protein component or antigenic component is an immunogenic component of the HCMV pentamer. Suitably, an antigenic or immunogenic component is a component capable of generating an immune response, such as an antibody response against such component, upon introduction into a subject such as a patient. Thus, for example, “the immunogenic component of HCMV pentamer” is a component capable of producing an anti-HCMV antibody response in a subject. Suitably, the immunogenic component comprises one or more (at least one) of the HCMV pentameric subunit components selected from gH, gL, pUL128, pUL130 and pUL131 (also known as pUL131A). In some embodiments, the immunogenic component comprises one or more of these “pUL” or “UL” components. In another embodiment, the immunogenic component comprises one or more of these gH or gL components. In one embodiment, the immunogenic component comprises a combination of one or more "UL" components and one or more components selected from gH or gL components. In another embodiment of the invention, the immunogenic component of the HCMV pentamer is an HCMV pentamer comprising all of the gH/gL/pUL128/pUL130/pUL131 subunits. Suitably, the gH/gL/pUL128/pUL130/pUL131 subunits are any of, including Towne (GI: 239909366), AD169 (GI: 219879600), Toledo (GI: 290564358) and Merlin (GI: 155573956). It has an amino acid sequence, or a functional equivalent thereof, corresponding to those derived from known HCMV strains (including both laboratory strains and/or clinical isolates). Functional equivalents refer to amino acid sequences that share some homology and differ in some amino acids, but retain functional properties capable of forming antigenic subunits or pentamers that provide a protective antibody. Suitable variants of the components gH/gL/pUL128/pUL130/pUL131A are described, for example, in WO2014/005959 (see pages 4 to 10), which is incorporated herein by reference. Advantageously, the use of HCMV pentameric subunits in a vaccine approach can provide immunogenic protection against infection from a wide range of HCMV virus strains due to the high level of homology between strains at the pentameric amino acid sequence level. .

In some embodiments, the antigenic component may correspond to a disease agent or component of a vector, or a portion thereof. For example, the antigenic component may lack a transmembrane domain for easy preparation. Suitably, in the HCMV pentamer, for example the immunogenic component of the HCMV pentamer, comprises a gH subunit having a truncated transmembrane domain (cleaved by deletion of one or more amino acids from this region), Subunits are secreted into the cell supernatant during protein production in host cells for easy purification.

In one embodiment, the gH/gL/pUL128/pUL130/pUL131A subunit has the amino acid sequence (or functional equivalent thereof) set forth in SEQ ID NOs: 28, 31, 35, 33, 36, respectively (with the indicated signal peptide. With or without). Functional equivalent refers to an amino acid sequence that shares some homology and differs in some amino acids, but retains functional properties capable of forming antigenic subunits or pentamers that provide a protective antibody. In some embodiments, functional equivalents may share 70%, 80%, 90% or more homology with the related amino acid sequence. In another embodiment, the gH/gL/pUL128/pUL130/pUL131A subunit is encoded by a nucleic acid sequence as set forth in SEQ ID NO: 13, 16, 20, 18, 21, or a codon optimized version thereof (signal peptide With or without the coding sequence for). In some embodiments, any one of the gH/gL/pUL128/pUL130/pUL131A subunits is a signal peptide, e.g., a signal peptide present in a native protein for that strain, a functional equivalent of the signal peptide, or HCMV. Signal peptides from other strains of. In some embodiments, any one of the gH/gL/pUL128/pUL130/pUL131A subunits may have a signal peptide derived from a heterologous protein. The choice of signal peptide can be determined to target the expressed protein to a specific cellular (or extracellular) location or to confer other functions. After expression of the subunit(s), the signal peptide can be enzymatically cleaved by natural cellular machinery or in vitro in the expression system used (eg, by signal peptidase). In some embodiments, any one of the gH/gL/pUL128/pUL130/pUL131A subunits may be expressed without a signal peptide. In some embodiments, native sequences comprising introns can be used where they can result in higher expression levels. Suitably, the native nucleic acid sequence for UL128 comprises two introns. In another embodiment, the nucleic acid sequence for UL131A comprises 1 intron. In some embodiments, the intron can be removed. In some embodiments, the native sequence can be codon-optimized for the relevant expression system.

In one embodiment of any aspect of the invention, the protein component or antigenic component is an immunogenic component of RSV virus, such as an adhesion glycoprotein (G protein) or a fusion glycoprotein (F protein), all of which are Control the steps. G is a highly glycosylated 90 kDa type II endogenous membrane protein, capable of mediating viral attachment to host cell membranes through interaction with heparan sulfate on proteoglycans, and is a good candidate for protein components.

The F protein is an intrinsic membrane protein composed of three F ₀ monomers that are processed during assembly into F ₁ and F ₂ subunits, which are covalently linked by two disulfide bonds. The F protein is highly conserved among RSV isolates from both A and B subgroups, and the amino acid sequence shows at least 90% identity. F is a 574 amino acid class I fusion protein consisting of a 50 kDa (kilodalton) carboxy-terminal F1 fragment and a 20 kDa amino-terminal F2 fragment; This is made into a trimer of a heterodimer. It is distinguished by two purine cleavage sites that release the 27 amino acid glycopeptide and expose the hydrophobic fusion peptide at the F1 amino terminus. There are two N-linked glycosylation sites in F2 and only one in F1. After removal of the 25 amino acid signal peptide and the 27 amino acid glycopeptide between F2 and F1, the remaining ectodomain of F consists of 472 amino acids. Only 25 amino acids in the F ectodomain differ between subtypes A and B.

To develop antigenic compositions from RSV-F protein, some studies have focused on preparing variants of the trimeric pre-fusion protein. Variants were prepared by gene fusion of two subunits of mature pre-F into a single chain. A DS-Cav1 variant in which F2 was gene fused to F1 and both the fusion peptide and the pep27 region were deleted was prepared. Since the difference in linkers between the F2 subunit and the F1 subunit appears to affect immunogenicity, the variant can select a variety of linkers. The native RSV-F protein sequence can be found in accession number P03420.1.

Several versions of the pre-fusion F protein have been studied and developed, and consequently published. All of these pre-fusion trimers may be suitable for use in the present invention. The DS-Cav1-stabilized fusion glycoprotein is derived from a natural protein. EP2222710, incorporated herein by reference, also discloses a recombinant RSV antigen comprising an F2 domain of an RSV-F protein polypeptide and a soluble F protein polypeptide comprising an Fl domain and a trimerization domain. Nat. Commun. 2015; 6: In 8143, Krarup et al , a highly stable pre-fusion RSV-F protein is described, which is incorporated by reference.

WO2014/160463, incorporated herein by reference, discloses isolated recombinant RSV-F protein stabilized in pre-fusion conformation as well as nucleic acid molecules encoding recombinant RSV-F protein.

WO2017/172890, incorporated herein by reference, discloses a substitution-modified pre-fusion RSV-F protein and nucleic acid encoding it. For further explanation, see Nat Struct Mol Biol. 2016 Sep; 23(9): 811-820, Iterative structure-based improvement of a respiratory syncytial virus fusion glycoprotein vaccine, M. Gordon Joyce, Baoshan Zhang, 　 Li Ou, 　 Man Chen, 　 Gwo-Yu Chuang, 　 Aliaksandr Druz, 　Wing-Pui Kong, Yen-Ting Lai, Stewart-Jones, Paul V. Thomas, 　 Joseph G. Van Galen, 　 Ulrich Baxa, 　 Kelly K. Lee, 　 John R. Mascola, 　 Barney S. Graham, 　 and Peter D. Kwong, and is also incorporated herein by reference.

Exemplary nucleic acid sequences encoding recombinant F ₂ -F ₁ ectodomain protomers linked to the T4 Fibritin trimerization domain are accession numbers: LP884611.1, LP884610.1, LP884609. 1 and LP884608.1.

In some embodiments, the protein or antigenic component may correspond to a disease agent or component of a vector, or a portion thereof. For example, the antigenic component may lack a transmembrane domain for easy preparation. Suitably, in the RSV-F protein or its pre-fusion conformation, for example, the immunogenic component of the F protein is an F with a truncated transmembrane domain (cleaved by deletion of one or more amino acids from this region). Including the ₂ -F ₁ subunit, the subunit is secreted into the cell supernatant during protein production in the host cell for easy purification. Therefore, the RSV-F protein lacks a functional TM domain. As an alternative, gene fusion with the first peptide tag can actually prevent the F protein from being present in the membrane despite the presence of a functional transmembrane domain.

In one embodiment, the pre-fusion stabilized subunits each have the amino acid sequence set forth in SEQ ID NOs: 50-58 (or functional equivalents thereof). Functional equivalent refers to an amino acid sequence that shares some homology and differs in some amino acids, but retains functional properties capable of forming, for example, antigenic subunits that provide a protective antibody. In some embodiments, functional equivalents may share 70%, 80%, 90% or more homology with the related amino acid sequence. In one embodiment, the pre-fusion stabilized RSV-F trimer may not include a heterotrimerization domain.

The protein component comprises a first peptide tag. The first peptide tag may be attached to a protein component by expressing the recombinant fusion protein. One of skill in the art would be aware of techniques for gene fusion of peptide sequences for expressing recombinant proteins in suitable cellular systems. For moieties comprising post-translational modifications such as glycosylation, it is preferred to express the recombinant protein in eukaryotic or mammalian cell lines.

Advantageously, the use of a first and a second peptide tag to form an isopeptide bond, such as the SpyTag-SpyCatcher system described herein, can be used to provide a protective/neutralizing/immunogenic effect (e.g., Anti-HCMV) large and/or multimer antigens, such as HCMV pentamers, or thereof, to moieties representing the large antigen, such as VLPs, in the correct formation and orientation so that the antigen is presented to the immune system in a manner capable of producing antibodies. Allows "decoration" of immunogenic components. Conventional vaccination approaches using soluble antigens (even large antigens such as multimer/5mer) may be less effective in exerting protective/neutralizing/immunogenic effects. Advantageously, when antigens (e.g. multimeric antigens) are presented on particles such as VLPs or nanoparticles, unlike soluble antigens, they represent geometric repeating arrangements of the same antigens that can strongly elicit an immune response. VLPs or other suitable particles of a larger size compared to the'free' antigen may also have a greater immunogenic effect. In addition, the display orientation of multimeric antigens such as HCMV pentamers can be important for immunogenicity. To attach a multimeric antigen to a particle, the use of paired tags such as the SpyTag-SpyCatcher system described herein allows the antigen to be attached to the particle in a particular advantageous orientation. For example, in the case of HCMV, the gH/gL subunit may be less likely to have a neutralizing epitope than the “UL” subunit. Thus, advantageously, the present invention provides HCMV on the particle by appropriate positioning of the first peptide tag, for example, so that the "UL" subunit is displayed towards the outside of the particle, and is therefore more readily available in the individual's immune system. The orientation of the pentamer is determined. As an alternative/additional, the position of the first peptide tag on the antigen can be determined to produce an orientation similar to that of the natural virus, thereby bringing the antigen into an orientation that is likely to elicit an immune response against the invading live virus. Present particles to the immune system.

In contrast, conventional approaches for presenting proteins to VLPs may involve chemical linkages, which means that these chemical reactions are more random, so that the correct (e.g., immunologically preferred) orientation of the antigen will be reliably obtained. It is not possible, and there is a disadvantage that only a small proportion of the linking reaction can be obtained. Moreover, processes involved in chemical conjugation may reduce the likelihood of maintaining the 3-D structure required for proper antigen presentation. Some drawbacks of conventional approaches are, for example, Brune et al. 2016; Scientific Reports , 6:19234, DOI: 10.1038/srep19234, Brune et al. Bioconjugate Chemistry, 2017, 28, 1544-1551, and Leneghan et al (2017) Scientific reports, 7:3811.

Similarly, gene fusion of antigens to viral coat proteins has proven to be challenging and time-consuming in determining the optimal expression conditions for both components and for misfolding problems. Moreover, gene fusion will not be suitable for the expression of large antigens or multi-component antigens as it is too difficult to achieve effective expression of the correct conformation.

In order to present the protein or antigenic component in an immunogenic manner, the position of the first peptide tag must be carefully designed so that the native protein conformation is maintained and, optionally, any post-translational modifications are properly maintained. For some antigens, maintaining glycosylation does not affect the way the epitopes are presented, but for other antigens, maintaining or removing them improves efficacy. In the case of a protein component that is a transmembrane protein, the transmembrane section of the protein component provides a good target for positioning the first peptide tag, and this sequence is not involved in the conformation of the antigenic protein component, e.g. Because it provides a role that is no longer needed in vaccines. If the protein component does not contain a transmembrane protein, it may be helpful to fuse the first peptide tag to the C- or N-terminus of the component or a subunit thereof (in the case of a multimer), but the first Peptide tags can also be included in any portion of the sequence. Alternatively, the first peptide tag can be placed in the loop of the protein or antigenic component.

In order to present the immunogenic component, such as the immunogenic component of the HCMV pentamer, the location of the first peptide tag must be carefully designed so that the native protein conformation is maintained. For HCMV, in one embodiment, it is attached through the gH subunit, suitably through the C-terminus of the gH subunit, or through the transmembrane domain (or a portion thereof) of the gH subunit. In addition to maintaining the conformation of the pentamer (or a component of the pentamer), this rational design also presents the target area of the pentamer towards the outside of the particle, as discussed above. As used herein, the target region is a part of a protein known to produce antibodies with a neutralizing effect, and may also be referred to as an immunogenic part.

To present the immunogenic component, such as the immunogenic component of the RSV pre-fusion F protein, the location of the first peptide tag must be carefully designed so that the native protein conformation is maintained. In the case of the RSV-F pre-fusion protein, in one embodiment, it is suitably attached through the C-terminus of the pre-fusion F, and through the 3′ end of the nucleic acid encoding it. In addition to maintaining the conformation of the pre-fusion F protein (or a component thereof), this rational design also presents most of the neutralizing epitopes of the pre-fusion F protein towards the outside of the particle, as discussed above. The above considerations apply equally to any other modification of the F protein. The inclusion of the first peptide tag at the C-terminus has been demonstrated by our studies, and the immunogenic protein component is correctly folded.

In one embodiment, the first and second peptide tags are part of a peptide tag/binding partner pair capable of forming an isopeptide bond. Such isopeptide binding may be spontaneous, i.e. no help is needed, or in need of assistance, i.e., assistance from a ligase or other helper. Suitably, the first and second peptide tags are SpyTag/SpyCatcher pairs. Suitably, the first and second peptide tags are SpyTag/SpyCatcher, SnoopTag/SnoopTagJr and SnoopCatcher; RrgATag/RrgATag2/DogTag and RrgACatcher, IsopepTag/IsopepTag-N and Pilin-C or Pilin-N, PsCsTag and PsCsCatcher; And SnoopTagJr and DogTag (mediated by SnoopLigase), and variants, derivatives and modifications of all of these systems.

Suitably, the first peptide tag is a peptide tag from a peptide tag/binding partner pair, such as SpyTag, and the second peptide tag is a binding partner, such as SpyCatcher. In another embodiment, the first peptide tag is a binding partner, such as SpyCatcher, and the second peptide tag is a peptide tag component from a peptide tag/binding partner pair, such as SpyTag.

Suitably, the first peptide tag is a peptide tag from a peptide tag/binding partner pair, such as SnoopTag, and the second peptide tag is a binding partner, such as SnoopCatcher. In another embodiment, the first peptide tag is a binding partner, such as SnoopCatcher, and the second peptide tag is a peptide tag component from a peptide tag/binding partner pair, such as SnoopTag. Thus, the first peptide tag is a “tag” or “catcher”; It can be seen that the second peptide tag may be a "catcher" or a "tag", respectively, which is a partner for this pair. Suitable peptide tag/binding partner pairs are described in detail in WO2011/09877, WO2016/193746, WO2018/18951 and WO2018/197854, which are incorporated herein by reference.

In one embodiment, the protein or antigenic component is attached to any one of SpyTag, SnoopTag, RrgATag, RrgATag2, DogTag, IsopepTag, IsopepTag-N, PsCsTag and SnoopTagJr as the first peptide tag.

The first peptide tag may be attached via a linker if necessary, which may be rigid or flexible. One of skill in the art will know which linker will be suitable.

In another embodiment, the moiety is attached to any one of SpyCatcher, SnoopCatcher, RrgACatcher, Pilin-C, Pilin-N, PsCsCatcher and DogTag (mediated by SnoopLigase) as a second peptide tag.

The moiety can be any suitable moiety previously discussed, including synthetic multimerization platforms.

The second peptide tag can be attached to any suitable position of the moiety, which does not affect the ability to fold and form the appropriate conformation. Gene fusion may be preferred. While it may be desirable to include a second peptide tag at the C- or N-terminus of the moiety, the second peptide tag may also be included in any part of the sequence. Alternatively, a second peptide tag can be placed in the loop of the moiety. For example, gene fusion of SpyCatcher to the N-terminus of the viral coat protein (CP3) of the RNA bacteriophage AP205 is described in Brune et al , Scientific Reports volume 6, Article number: 19234 (2016). Alternative fusion using self-assembled synthetic proteins as a multimerization platform is discussed in Bruun et al , ACS Nano, 2018, 12 (9), pp 8855-8866. The second peptide tag may alternatively be attached via chemical conjugation.

The second peptide tag may be attached via a linker if necessary, which may be rigid or flexible. One of skill in the art will know which linker will be suitable.

In one embodiment, the antigenic component, such as HCMV pentamer or immunogenic component thereof, is attached to SpyTag. A suitable SpyTag has the amino acid sequence set forth in SEQ ID NO: 30.

The SpyTag may be attached through a linker. Suitable linkers include linkers having the amino acid sequence set forth in SEQ ID NO: 29.

In another embodiment, the moiety is attached to the SpyCatcher binding partner (second peptide tag). The moiety may suitably be HBsAg. A suitable SpyCatcher has the amino acid sequence set forth in SEQ ID NO: 38. In one embodiment, SpyCatcher is attached via a linker. The linker may be a rigid linker or a flexible linker, suitably the linker has the amino acid sequence set forth in SEQ ID NO: 39.

In another embodiment, the protein composition or antigenic composition according to any aspect or embodiment of the invention further comprises another, preferably different, protein comprising the first peptide tag.

In another embodiment, the composition according to any aspect or embodiment of the invention further comprises another, preferably different, antigen comprising a first peptide tag, such as another HCMV antigen. Suitably, the other HCMV antigen is glycoprotein B. Suitably, the glycoprotein B sequence is described for example in WO2014/005959, see SEQ ID NO: 21, 22, 23 or 36. In one embodiment, the composition comprises particles (eg, VLPs) that represent both HCMV pentamers and other HCMV antigens.

In one embodiment, the composition is an immunogenic composition or a vaccine composition. Preferably, the immunogenic or vaccine composition is one capable of inducing an immune response such as an antibody response when administered to an individual. Suitably, the immune response may be a protective immune response. Suitable immunogenic compositions may further comprise additional ingredients including adjuvants, immunostimulants and/or pharmaceutically acceptable excipients.

Suitable adjuvants are for example aluminum, peptides, can be as squalane, liposomes, water type based on the emulsion and saponins, Alhydrogel ^®, MF59, AS01, MatrixM, unevenness wheat dipep sulfide (muramyl dipepide) and comprise a Quil A I can. In addition, water-in-oil adjuvants are suitable. Squalene-Oil-in-water emulsions such as Addavax ^™ are suitable.

Thus, in another aspect or embodiment of the invention, an immunogenic or vaccine composition comprising a composition according to the invention is provided. Suitably, the vaccine composition comprises a vaccine dose, which is an amount of the composition according to the invention that provides an immunogenic, preferably immunoprotective effect from an infectious agent/vector, such as a neutralizing effect from HCMV infection. Suitably, the vaccine composition comprises a vaccine dose, which is an amount of the composition according to the invention, which provides a neutralizing effect from an infectious agent/vector, such as a neutralizing effect from RSV infection. Antibodies, preferably neutralizing antibodies, generated against an immunogenic composition can be detected and measured by methods familiar to those of skill in the art, including, for example, standardized ELISA assays or microneutralization assays as described herein.

In another aspect, a VLP is provided comprising:

i) a moiety comprising a first peptide tag

ii) a protein comprising a second peptide tag

The first peptide tag and the second peptide tag form an isopeptide bond. In some embodiments, the moiety is HBsAg. However, as described above, any suitable moiety may be used.

Suitably, the first peptide tag is a peptide tag from a peptide tag/binding partner pair, such as SpyTag, and the second peptide tag is a binding partner, such as SpyCatcher. In another embodiment, the first peptide tag is a binding partner, such as SpyCatcher, and the second peptide tag is a peptide tag from a peptide tag/binding partner pair, such as SpyTag. Other suitable peptide tag/binding partner pairs are described herein and are known to those of skill in the art. Suitably, the first and second peptide tags are SpyTag/SpyCatcher, SnoopTag/SnoopTagJr and SnoopCatcher; RrgATag/RrgATag2/DogTag and RrgACatcher, IsopepTag/IsopepTag-N and Pilin-C or Pilin-N, PsCsTag and PsCsCatcher; And SnoopTagJr and DogTag (mediated by SnoopLigase), and variants, derivatives and modifications of all of these systems.

Suitably, the protein comprising the second peptide tag is a protein or peptide complex exceeding 50 kDa. Proteins containing the second peptide tag are 50 kDa, 60 kDa, 70 kDa, 80 kDa, 90 kDa, 100 kDa, 110 kDa, 120 kDa, 130 kDa, 140 kDa, 150 kDa or 160 kDa, 170 kDa, 180 kDa, 190 kDa or greater, such as greater than 200 kDa, greater than 300 kDa, or greater than 400 kDa.

In one embodiment, the protein comprising the second peptide tag is a multimeric protein. In one embodiment, the protein comprising the second peptide tag is an antigen, preferably a multimeric antigen. Suitably, the multimeric antigen may be the HCMV pentamer described herein. Suitably, the protein may be an RSV-F protein or a derivative thereof (eg, pre-fusion F protein). In one embodiment, the protein comprising the second peptide tag is an immunogenic component of the HCMV pentamer. The HCMV pentamer (gH/gL/pUL128/pUL130/pUL131) described herein and comprising a suitable linker and tag has a molecular weight of greater than 160 kDa. Other suitable large or multimeric proteins or antigens include antigens from other infectious agents, including viruses such as influenza virus, RSV, and the like.

Advantageously, the use of HBsAg as carrier (VLP) in this manner has the potential to produce an anti-HepB boost, described as an anti-hepatitis B virus (HBV) reaction as an alternative.

In another aspect, a VLP is provided comprising:

i) a protein comprising the first peptide tag

ii) a moiety comprising a second peptide tag

The first peptide tag and the second peptide tag form an isopeptide bond. In some embodiments, the moiety is HBsAg. However, as described above, any suitable moiety may be used.

Suitably, the first peptide tag is a peptide tag from a peptide tag/binding partner pair, such as SpyTag, and the second peptide tag is a binding partner, such as SpyCatcher. In another embodiment, the first peptide tag is a binding partner, such as SpyCatcher, and the second peptide tag is a peptide tag from a peptide tag/binding partner pair, such as SpyTag. Other suitable peptide tag/binding partner pairs are described herein and are known to those of skill in the art. Suitably, the first and second peptide tags are SpyTag/SpyCatcher, SnoopTag/SnoopTagJr and SnoopCatcher; RrgATag/RrgATag2/DogTag and RrgACatcher, IsopepTag/IsopepTag-N and Pilin-C or Pilin-N, PsCsTag and PsCsCatcher; And SnoopTagJr and DogTag (mediated by SnoopLigase), and variants, derivatives and modifications of all of these systems.

Suitably, the protein comprising the first peptide tag is a protein or peptide complex that exceeds 50 kDa. The protein comprising the first peptide tag is 50 kDa, 60 kDa, 70 kDa, 80 kDa, 90 kDa, 100 kDa, 110 kDa, 120 kDa, 130 kDa, 140 kDa, 150 kDa or more than 160 kDa, especially 200 kDa , 300 kDa or even more than 400 kDa protein or peptide complex. In one embodiment, the protein comprising the first peptide tag is a multimeric protein. In one embodiment, the protein comprising the second peptide tag is an antigen, preferably a multimeric antigen. Suitably, the multimeric antigen may be the HCMV pentamer described herein. Suitably, the protein may be an RSV-F protein or a derivative thereof (eg, pre-fusion F protein). In one embodiment, the protein comprising the first peptide tag is an immunogenic component of the HCMV pentamer. HCMV pentamers (gH/gL/pUL128/pUL130/pUL131A) described herein and comprising a suitable linker and tag have a molecular weight of greater than 160 kDa. Other suitable large or multimeric proteins or antigens include antigens from other infectious agents, including viruses such as influenza virus, RSV, and the like.

Advantageously, using HBsAg as a carrier (VLP) in this way also has the potential to produce an anti-HBV boost.

In another aspect of the invention, there is provided an HCMV pentamer linked to SpyTag as described herein.

According to another aspect of the present invention, there is provided a method for preparing a composition or VLP according to the present invention, the method comprising the steps of:

-Introducing into a first host cell a first nucleic acid encoding a first gene fusion of a first protein to a first peptide tag;

-Incubating said first host cell under conditions expressing said first gene fusion; Optionally purifying the expressed component;

-Introducing a second nucleic acid encoding a second gene fusion of a second protein to a second peptide tag into a second host cell;

-Incubating the second host cell under conditions expressing the second gene fusion; Optionally purifying the expressed component;

-Incubating the expressed components under conditions that form an isopeptide bond between the first and second peptide tags; Optionally purifying the obtained composition.

Suitably, the expressed components are incubated together to form an isopeptide bond. The formation of the isopeptide bond may require co-incubation with a ligase or analog.

Suitably, the composition according to the present invention or a method of preparing a VLP may be for preparing a composition comprising the antigenic component shown in the VLP.

In some embodiments, when the "immunogenic component of HCMV pentamer" comprises the entire HCMV pentamer, the recombinant production of the component of the HCMV pentamer will be expressed with the correct stoichiometry for the pentamer from which each subunit will be formed. In addition, they need to be folded correctly for assembly. In this embodiment, only some of the complexes of the necessary pentamers (e.g., gH/gL dimers and tetramers, or tetramers lacking any of the 5 subunits) need to be excluded from the final product. Advantageously, the present invention can be linked by expressing all vaccine components (i.e., 5 subunits of HCMV pentamer and HBsAg) in one system by providing a simple approach to preparing the components individually and then conjugating them. Overcome existing problems. Thus, in one embodiment, the purification tag is incorporated into UL130 (Hofmann et al, DOI 10.1002/bit 25670). Similar principles can be applied to other immunogenic components.

In some embodiments, when the “immunogenic component of RSV-F protein” comprises the entire F protein or a derivative thereof, the recombinant production of the component of the F protein or derivative thereof is a derivative comprising a pre-fusion F protein trimer. Together, it must be correctly folded for assembly.

Suitably, the method is for preparing a composition comprising the HCMV pentamer shown in the HBsAg VLP. Suitably, the method is for preparing a composition comprising the RSV-F pre-fusion F protein trimer shown in the HBsAg VLP.

In another aspect of the invention, a vaccine is provided for use in the prevention and/or treatment of a disease. Suitably, the vaccine comprises a composition or VLP according to any aspect or embodiment of the invention. In one embodiment, the disease is an HCMV infection. In another aspect, a method of prophylactic treatment for HCMV is provided. Suitably, the vaccine is for use in humans. Suitably, the vaccine is for use in adult humans, for example women of childbearing age or pregnant women. In another aspect, the present invention provides a method for inducing an immunogenic response, e.g., a protective immune response, to HCMV in an individual, the method comprising administering a composition according to any aspect or embodiment of the present invention. Include.

In another aspect of the invention, a composition according to any aspect of the invention for use as a medicament is provided.

In a further aspect of the invention there is provided a composition according to any aspect of the invention for use as a vaccine, preferably as a vaccine for use in the prevention and/or treatment of HCMV infection. Compositions for use as medicaments or vaccines according to the invention can be administered to human adults, for example to women of childbearing age or to pregnant women.

In another aspect, the invention provides a nucleic acid molecule for use in a method according to the invention. In one embodiment, the nucleic acid molecule according to the invention comprises a nucleic acid sequence encoding the amino acid sequence set forth in any one of SEQ ID NOs: 27 to 41. In one embodiment, the nucleic acid molecule according to the invention comprises a nucleic acid sequence set forth in any one of SEQ ID NOs: 12-26 or 42-46.

In another aspect, the invention provides a plurality of nucleic acid molecules comprising a nucleic acid molecule encoding the amino acid sequence set forth in SEQ ID NOs: 27-41. In one embodiment, the nucleic acid molecules of the present invention include those having the sequence set forth in any one of SEQ ID NOs: 12-26 or 42-46.

In another aspect, the invention provides a nucleic acid molecule for use in a method according to the invention. In one embodiment, the nucleic acid molecule according to the invention comprises a nucleic acid sequence encoding the amino acid sequence set forth in any one of SEQ ID NOs: 50 to 58. In one embodiment, the nucleic acid molecule according to the invention comprises the nucleic acid sequence set forth in any one of SEQ ID NOs: 47 to 55.

In another aspect, the invention provides a plurality of nucleic acid molecules comprising a nucleic acid molecule encoding the amino acid sequence set forth in SEQ ID NOs: 50-58. In one embodiment, the nucleic acid molecules of the invention comprise those having the sequence set forth in any one of SEQ ID NOs: 47-55.

In another aspect, the invention provides a nucleic acid molecule according to the invention or a vector comprising a plurality of nucleic acid molecules. Suitably, the vector is an expression vector for expressing the amino acid sequence of any component of the composition according to the invention.

In another aspect, the invention provides a host cell for expressing the components of the composition according to the invention. Suitable host cells may be for transient or stable expression of these components. Methods and host cells for expressing CMV proteins are described, for example, in WO2014/005959 and WO2016/067239, all of which are incorporated by reference. In some embodiments, the component may be glycosylated.

In another aspect of the invention, a kit is provided comprising a composition according to the invention for use in a prime-boost vaccination regime. Suitably, the kit may comprise a prime composition comprising a first immunogenic composition according to the present invention and a boost composition comprising a second immunogenic composition according to the present invention. Alternatively, the kit can be provided to provide a single or multiple dose vaccination regimen, i.e. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 doses. Thus, in another aspect, the present invention provides a dosage regimen comprising doses applied approximately 3 weeks apart.

Figure 1: SDS-PAGE and Western blot analysis of pentameric-SpyTag purified under non-reducing and reducing conditions. Lane 1: ColorPlus Prestained Broad Range Protein Ladder, size expressed in kDa. Lane 2: non-reducing sample; Lane 3: reduction sample. A) SDS-PAGE and Coomassie staining analysis, the location of the HCMV pentameric component is indicated, non-reduced to the left of the gel, reduced to the right. B) Western blot analysis using anti-HCMV pentameric antibody.
Figure 2: SDS-PAGE and Western blot analysis of purified SpyCatcher-HBsAg under non-reducing (NR) and reducing conditions (R). A) SDS-PAGE and Coomassie staining analysis. B) Western blot analysis using anti-HBsAg monoclonal antibody.
Figure 3: HPLC analysis using a s200increase 3.2/300 column. A) 10 μl of purified HCMV pentamer-SpyTag was loaded and eluted as a single peak. B) 10 μl of purified SpyCatcher-HBsAg was loaded and eluted as a single major peak in the pore volume of the column.
Figure 4: SDS-PAGE and Western blot analysis of pentameric-SpyTag and SpyCatcher-HBsAg conjugated under reducing conditions. 1: ColorPlus Prestained Broad Range Protein Ladder, size expressed in kDa. 2: conjugation; 3: Pentameric-SpyTag; 4: SpyCatcher-HBsAg. A) SDS-PAGE and Coomassie staining analysis. B) Western blot using anti-HBsAg monoclonal antibody. C) Western blot using anti-5mer polyclonal antibody.
Figure 5: HPLC analysis using s200 increase 3.2/300 column. 30 μl of the conjugated pentamer-SpyTag-SpyCatcher-HBsAg was loaded and eluted as the main peak in the pore volume of the column.
Figure 6: Immunogenicity of HCMV pentameric-HBsAg vaccine versus pentameric protein vaccine, adjuvanted with Addavax after a single immunization. BALB/c mice were immunized with 1 μg or 0.1 μg of HCMV pentameric-SpyTag as pentameric-HBsAg VLP or as soluble protein. Titres were measured by standardized ELISA from mouse serum. Lines represent mean and error bars represent standard deviation (n = 10). Mice immunized with HCMV pentameric-HBsAg VLPs exhibit a substantially stronger serum IgG antibody response compared to mice immunized with HCMV pentameric protein alone, even though the pentameric-equivalent VLP dose is 10-fold lower.
Figure 7: Neutralising activity in mouse serum immunized with HCMV pentameric-HBsAg vaccine compared to pentameric protein vaccine. The vaccine had Addavax as an adjuvant and responded after one immunization (after prime) or two immunizations (after boost). NT ₅₀ was measured in ARPE-19 cells infected with AD169 ^wt131 strain (representing a functional pentamer). The same assay shows neutralizing titers for Cytogam and commercially available neutralizing anti-gH mAb (HCMV16 (51C1) from Bio-Rad Antibodies).
Figure 8: Immunogenicity of HCMV pentameric-HBsAg vaccine versus pentameric protein vaccine, without adjuvant, after one or two immunizations. BALB/c mice were immunized with 1 μg or 0.1 μg of HCMV pentameric-SpyTag conjugated to SpyCatcher-HBsAg (pentameric-HBsAg), or 1 μg of pentameric-SpyTag protein. Titer was determined by standardized ELISA from mouse serum. Lines represent mean and error bars represent standard deviation (n = 10). Mice immunized with HCMV pentameric-HBsAg VLPs exhibit a substantially stronger serum IgG antibody response compared to mice immunized with HCMV pentameric protein alone, even though the pentameric-equivalent VLP dose is 10-fold lower.
Figure 9: Neutralizing activity in mouse serum immunized with HCMV pentameric-HBsAg vaccine compared to pentameric protein vaccine. The vaccine did not use an adjuvant and showed a response after one immunization (after prime) or two immunizations (after boost). NT50 was measured in ARPE-19 cells infected with AD169 ^wt131 strain (representing a functional pentamer). The same assay shows neutralizing titers for Cytogam and commercially available neutralizing anti-gH mAb (HCMV16 (51C1) from Bio-Rad Antibodies).
Figure 10: SDS-PAGE and Western blot analysis of purified RSV-F-SpyTag under non-reducing and reducing conditions. A) SDS-PAGE and Coomassie staining analysis, lane 1: ColorPlus Prestained Broad Range Protein Ladder; Lane 2: non-reducing sample; Lane 3: reduction sample. B) Western blot analysis using anti-RSV-F monoclonal antibody, lane 1: ColorPlus Prestained Broad Range Protein Ladder; Lane 2: non-reducing sample; Lane 3: reduction sample.
Figure 11: SDS-PAGE and Western blot analysis of RSV-F-SpyTag conjugated with SpyCatcher-HBsAg under reducing conditions. 1: ColorPlus Prestained Broad Range Protein Ladder, 2: RSV-F-SpyTag--SpyCatcher-HBsAg conjugate, 3: RSV-F-SpyTag, 4: SpyCatcher-HBsAg. A) SDS-PAGE and Coomassie staining analysis. B) Western blot using anti-HBsAg monoclonal antibody. C) Western blot using anti-RSV-F monoclonal antibody.
Figure 12: Immunogenicity of conjugated RSV-F-SpyTag--SpyCatcher-HBsAg ('Sc9-10-HBsAg') versus unconjugated RSV-F-SpyTag ('Sc9-10'). BALB/c mice, 1 μg of RSV-F-SpyTag or RSV-F-SpyTag protein conjugated to SpyCatcher-HBsAg (RSV-F VLP) with or without Addavax™ (n = 8) as an adjuvant Was immunized with 1 μg of. The RSV-F antigen is sc9-10 DS-Cav1 A149C Y458C-SpyTag.

Virus-like particles

Conventionally, the vaccine approach has used whole pathogens that have been attenuated or killed, but this has been replaced by using recombinant subunit vaccines containing proteins from appropriate pathogens. Recently, an approach using virus-like particles (VLP) has been developed. VLPs are particles in their size (approximately 20-200 nm), morphology and repetitive protein arrangement similar to viruses, but lacking any genetic material from pathogens. Because of its size, VLPs are more likely to excrete into the lymph nodes, making them ideal for uptake and presentation by antigen-presenting cells. In addition, their repetitive structure promotes complement fixation and B cell receptor cross-linking (Kushnir et al. Vaccine 2012; Vol 31(1):58-83). However, their mechanism of action is not limited to theory.

HCMV

Human cytomegalovirus (HCMV, also known as human herpesvirus-5 (HHV-5)) is a virus to which most adults are exposed, and the initial infection is usually mild or asymptomatic. After infection, the virus remains latent in the body, but can cause serious illness in immunocompromised patients or the elderly. HCMV is the leading cause of birth defects in developed countries. Up to 4 out of 200 infants are born with HCMV from congenital infections, with up to 10% of these having long-term consequences. HCMV infection is also associated with hypertension and atherosclerosis in adults (Cheng et al. (May 2009). Fruh K, ed. "Cytomegalovirus infection causes an increase of arterial blood pressure". PLoS Pathog. 5 (5): e1000427).

The pentameric complex of HCMV containing the viral protein gH/gL/pUL128/pUL130/pUL131A can reduce the risk of HCMV transmission perinatally as well as neutralize the virus that has been introduced into the epithelial cells by antibodies against this complex. Based on the observation that there is, it has been identified as a potentially useful vaccine target for HCMV. However, despite intensive efforts, no successful HCMV vaccine has been developed to date.

HCMV Pentamer

HCMV strains, including clinical isolates and laboratory strains, differ in their genomic sequence. HCMV strains include Merlin (GI:155573956), Towne (GI239909366) and AD169 (GI:219879600), Toledo (GI290564358) and TB40/E. HCMV contains a number of membrane proteins and protein complexes. The pentameric protein gH/gL/pUL128/pUL130/pUL131A is important for HCMV infection of epithelial and endothelial cells, thought to be through endocytic pathways. Other combinations of these complex components have been shown to be important for infection of fibroblasts, for example. the “pUL” subunit/component is also referred to as “UL”; “pUL131” is also referred to as “pUL131A” and “pUL131a” or “UL131A”.

Various HCMV strains have been deposited with ATCC and can be found as Merlin (VR-1590), Towne (VR-977) and AD169 (VR-538). Genomic sequences can be referenced through accession numbers: Merlin (AY446894.2), Towne (GO121041.1), AD169 (FJ527563.1), Toledo (GU37742.2) and TB40/E (KF297339.1)

RSV

Respiratory syncytial virus is the leading cause of serious respiratory disease in infants worldwide. About 3.4 million infants under 5 years of age are hospitalized every year for severe RSV lower respiratory tract infection, and the incidence rate is highest in infants under 6 months of age. Most deaths occur in infants under the age of one and in developing countries. At present, prevention and management options are limited.

RSV -F Fusion-Before Trimer

F glycoprotein is a type I virus fusion protein. It is believed that the RSV F precursor (F0) is cleaved by a purine-like protease at two sites, resulting in three fragments. The shorter N-terminal fragment (F ₂ ) is covalently attached to the larger C-terminal fragment (F ₁ ) by two disulfide bonds. Intervening fragments of 27 amino acids dissociate after cleavage and are not found in mature proteins.

As previously discussed, a number of stabilized pre-fusion F trimers can be used. In the examples presented herein, exemplary sequences encoding these pre-fusion trimers can be found as SEQ ID NOs: 48, 48, 54 and 55. Sequences comprising fusion with SpyTag are included as SEQ ID NOs: 47 and 53. The amino acid sequence is shown as SEQ ID NOs: 51, 52, 57 and 58 for the pre-fusion trimer and SEQ ID NOs: 50 and 56 for SpyTag. Other exemplified sequences are mentioned herein.

Peptide Tag/Binding Partner Pair

Proteins capable of spontaneously forming isopeptide bonds (so-called “isopeptide proteins”) are peptide tag/polypeptide binding partner pairs (ie, two-part linkers) that are covalently bonded to each other to provide an irreversible interaction. ) (See, for example, WO2011/098772 and WO 2016/193746, incorporated herein by reference, and WO2018/189517 and WO2018/197854, which are incorporated herein by reference). In this regard, a protein capable of spontaneously forming an isopeptide bond can be expressed as a separate fragment, thereby providing a peptide tag and a polypeptide binding partner for the peptide tag, wherein the two fragments form an isopeptide bond. Covalently reconstituted by, thereby linking molecules or components fused to the peptide tag and its polypeptide binding partner. The isopeptide bonds formed by the peptide tag and its polypeptide binding partner are subject to non-covalent interactions, e.g., at high temperatures (up to 95° C.) over a long period (e.g., several weeks), with strong force or harsh chemical treatment (e.g. : Stable under conditions that rapidly dissociate in pH 2-11, organic solvents, detergents or denaturants).

An isopeptide bond is an amide bond formed between a carboxyl/carboxamide group and an amino group, wherein at least one of the carboxyl group or amino group is outside the protein main chain (backbone of the protein). These bonds are chemically irreversible under typical biological conditions, and they are resistant to most proteases. As isopeptide bonds are essentially covalent bonds, they induce some of the most strongly measured protein-protein interactions.

Briefly, a two-part linker, i.e. a peptide tag and its polypeptide binding partner (so-called peptide tag/binding partner pair) can be derived from a protein (isopeptide protein) capable of spontaneously forming an isopeptide bond, and the protein The domains of are individually expressed and a peptide “tag” comprising one of the residues involved in isopeptide binding (eg, aspartate or asparagine, or lysine) and other residues involved in isopeptide bonding (eg, lysine, Or aspartate or asparagine) and a peptide or polypeptide binding partner (or “catcher”) comprising at least one other moiety (eg glutamate) necessary to form an isopeptide bond. When the peptide tag and the binding partner are mixed, an isopeptide bond is spontaneously formed between the tag and the binding partner. Thus, by individually incorporating the peptide tag and binding partner into different molecules or components, such as proteins, covalently linking the molecules or components together through an isopeptide bond formed between the peptide tag and binding partner, That is, it is possible to form a linker between molecules or components by incorporating the peptide tag and a binding partner.

The spontaneous formation of the isopeptide bond can be isolated and there is no need to add any other entities. Some peptide tags and tag partner pairs may require the presence of a helper entity such as a ligase to generate an isopeptide bond.

SpyTag / SpyCatcher said peptide tag / binding partner pair (two-part linker) is streptococcus blood comes Ness (Streptococcus pyogenes) was derived from a domain of CnaB2 FbaB protein (Zakeri et al., 2012, Proc Natl Acad Sci USA 109 , E690-697), and has been used in various applications including vaccine development (Brune et al., 2016, Scientific reports 6, 19234; Thrane et al., 2016, Journal of Nanobiotechnology 14, 30).

Suitably, the first and second peptide tags form a peptide tag/binding pair called SpyTag/SpyCatcher. Suitably, the components are SpyCatcher DeltaN1 (ΔN1) SpyCatcher and (Li, L., Fierer, JO, Rapoport, TA & Howarth, M. Structural analysis and optimization of the covalent association between a peptide and SpyCatcher Tag. J. Mol .. Biol 426, as the same) as described in 309-317 (2014), which "SpyCatcher" (SEQ ID NO: 23 has an amino acid at the N- terminus cutting as compared with 38).

In another embodiment, the first and second peptide tags are a mutated version of SpyTag/SpyCatcher that exhibits increased kinetics for isopeptide bond formation, e.g. as described in co-pending application GB1706430.4. Form a tag/bond pair. In some embodiments, this mutated form is the attachment of a large protein (e.g., >50 kDa or >100 kDa, such as the >160 kDa HCMV pentameric protein described herein), and/or when a slow response or steric hindrance may occur. May be useful for

In another embodiment, the isopeptide protein may comprise SnoopTag/SnoopCatcher, for example described in WO 2016/193746.

In some embodiments, one or both of the isopeptide proteins may have an N- or C-terminal truncation while still maintaining the reactivity of the isopeptide bond.

Illustrative first and second peptide tag pairs (peptide tag/binding partner pair; reactive pair) are described in the table below:

For example, WO2011/098772, WO2016/193746, GB1706430.4, GB 1705750.6 or Li, L., et al., J. Mol. Biol. 426, 309-317 (2014).

Variants, derivatives and modifications of the above binding pairs can be prepared by any suitable means. Variants, derivatives and functionally acting modifications may include amino acid additions, substitutions, alterations or deletions that retain the same function with respect to the ability to form isopeptide bonds with the relevant binding partner.

For some binding pairs, mediation by a third entity such as an enzyme is required. For example, SnoopLigase can be used to mediate the formation of bonds between SnoopTagJr and DogTag. Therefore, the help of enzymes such as ligase may be required for pairing.

HBsAg

"HBsAg" refers to a surface antigen or a portion thereof from hepatitis B virus (HBsAg). In one embodiment, HBsAg may refer to the N-terminus of HBsAg, such as the HBsAg sequence set forth in SEQ ID NO: 41, comprising 226 amino acids of the S protein of hepatitis B virus (adw serotype). Suitably, the HBsAg comprises Pro Val Thr Asn, a four amino acid sequence representing the four carboxy terminal residues of the hepatitis B virus (adw serotype) preS2 protein, and Valenzuela et al., (1979)'Nucleotide sequence. of the gene coding for the major protein of hepatitis B virus surface antigen' Nature 280:815-819. VLPs formed from HBsAg have been approved for clinical use against hepatitis B (Kushnir et al. Vaccine 2012; Vol 31(1):58-83), Recombivax HB (https://vaccines.procon.org/sourcefiles/) recombivax_package_insert.pdf), and Energix B (https://au.gsk.com/media/217195/engerix-b_pi_006_approved.pdf). HBsAg has also been used as the basis for the pre-erythrocytic malaria vaccine RTS,S, which has completed phase 3 clinical trials and is the most advanced malaria vaccine to date (http://www. malariavaccine.org/sites/www.malariavaccine.org/files/content/page/files/RTSS%20FAQs_FINAL.pdf; Kaslow and Biernaux, Vaccine 2015, Vol. 33(52): 7425-7432).

Linker details

The distance between proteins (e.g., VLPs and decorative antigens) can affect the availability of antigenic epitopes in the protein, stability of the protein/s, and also by accessibility of one of the isopeptide binding partners (e.g. SpyTag/SpyCatcher). Due to this, the bonding efficiency may also be affected. Therefore, linkers with appropriate properties can be selected to optimize availability, stability and/or accessibility. Linkers can be broadly subdivided into flexible and rigid subtypes.

Flexible Linker

When the linked domains need to be moved, a flexible linker can be used. Generally they are composed of small non-polar (eg Gly) or polar (eg Ser, Thr) amino acids, and their small size provides flexibility (Chen et al., 2013 Adv Drug Deliv Rev. Oct 15; 65 (10): 1357-1369). The addition of Ser or Thr can help to maintain the stability of the solution, and adjusting the length can affect the proper folding of the protein (Chen et al., 2013). Any suitable flexible linker having properties and lengths suitable for the entity concerned may be used. Suitably, the flexible linker may comprise a combination between 2 to 70 amino acids of this type.

Solid linker

In some cases, rigid linkers may be preferred, as they may help to provide protein separation. The rigid linker has a secondary structure. One of the most common rigid linkers is (EAAAK) _n (where n is the number of repetitions), which adopts an α-helix structure (Arai et al., (2001) Protein Eng. Aug;14(8):529-32) . Other rigid linkers may comprise a proline rich sequence such as (XP) _n , where X is any amino acid but preferentially Ala (A), Lys (K) or Glu (E), where proline is the conformational form Provides a conformal constraint (Chen at al., 2013).

Other suitable linkers are, for example, Klein et al. (2014) Protein Eng Des Sel. Oct; 27(10): 325-330. Any suitable rigid linker with properties and lengths suitable for the entity concerned may be used. Suitably, the rigid linker may comprise a combination between 2 to 70 amino acids of this type.

Host cell and expression vector

Host cells suitable for the expression of nucleic acids to prepare proteins and compositions according to the present invention are known to those skilled in the art.

In one embodiment, the host cell will be suitable for transient expression. In other embodiments, the host cell will be a cell capable of forming a stable cell line. Suitably, coding sequences encoding antigenic components such as HCMV pentamer and RSV-F protein, including peptide tags that form isopeptide bonds, will be incorporated into one host cell. In one embodiment, each nucleic acid sequence encoding a multimeric subunit, such as a pentameric, is contained in a different plasmid/vector, for example, by transfecting a host cell with all five plasmids/vectors and culturing it under appropriate conditions. When doing so will induce the production of pentamers by the host cell. In other embodiments, the plasmid/vector may comprise a combination of one or more coding sequences such that at least 1, 2, 3, 4 or 5 plasmids can be introduced. Alternatively, the entire fusion peptide coding sequence can be provided in one vector such that the entire protein component and the first peptide tag are encoded in the same vector.

In one embodiment, these vectors are used to stably integrate the coding sequence into the genome of the host cell. Host cells suitable for stable expression include mammalian cells, such as rodent cells, including HEK cells (human embryonic kidney 293 cells) or Chinese Hamster Ovary (CHO) cells. Mammalian cells and vectors suitable for expression of the protein components of the compositions according to the invention are known to those skilled in the art, for example WO2016/067239, pages 15-16 and Hofmann et al., (2015) Biotech and Bioeng, 112(12) :2505-2515. Stable construct sequences exemplified for the expression of the components according to the invention can be found in Example 3 below.

Affinity purification

In some embodiments, such expression constructs for use in expressing components of a composition according to the invention may comprise a “tag” sequence or sequences that facilitate purification, such as affinity purification. Any suitable tag, such as an affinity tag, can be included to separate the protein component and the first peptide tag from the resulting system. Those skilled in the art of producing recombinant proteins are aware of systems such as His-tag and Strep-tag that may be included for purification purposes. Such tags are preferred because they dramatically aid in protein purification and have little detrimental effect on biological or biochemical activity. Suitable tag sequences include C-tag, histidine tag (His-tag), streptavidin tag (Strep-tag), maltose-binding protein (MBP), glutathione-S-transferase (GST) and FLAG tag. do.

All of the protein components and/or moieties may comprise an affinity purification tag. For ease of use, they are generally genetically fused at the C- or N-terminus of the protein.

Therefore, in some embodiments, for example, the gH, gL, pUL128, pUL130, pUL131A (or fragment thereof) subunit of HCMV, the RSV pre-fusion F protein, or the HBsAg peptide/protein is added at the N- or C-terminus. May contain amino acid residues of, which facilitates purification. Such additional amino acid residues may include tags such as, for example, His-tags or C-tags. In some embodiments, C-tags can provide cleaner tablets. Other suitable tag sequences include maltose-binding protein (MBP), Strep-tag, glutathione-S-transferase (GST) and FLAG tag. In some embodiments, the tag can be linked to an amino acid sequence in a manner that can be cleaved after purification, for example using a cleavable linker. In other embodiments, non-affinity purification methods can be used.

In another embodiment, the RSV pre-fusion F protein may comprise an additional amino acid residue at the C- or N-terminus, which facilitates purification. The RSV pre-fusion F protein exemplified herein has a C-tag for affinity purification.

Conjugation of the first and second peptide tag pair

The conjugation of the first and second peptide tag/binding partner/reactive pair may be performed overnight at 4°C. As an alternative, the conjugation reaction can be carried out for 3-4 hours at room temperature since the coupling rate is expected to increase at room temperature. The optimal ratio of the first and second binding partners for a given coupling reaction depends on the size of each binding partner. For example, a 1:1.5 molar ratio of VLP monomer to antigen may be sufficient for smaller antigens (~20 kDa), whereas a 1:1 mass ratio in combination with the same VLP monomer. For larger antigens (> 100 kDa) it may be sufficient. However, all of the proportions result in excess antigen (smaller binding partners). Any excess antigen can be removed, for example by size exclusion chromatography (SEC) or dialysis. Dialysis may be more suitable for smaller antigens because it is not as effective as SEC. Alternatively, the ratio of VLP/particle to antigen can be optimized so that all antigens are conjugated and downstream purification is not required. A suitable final protein concentration of approximately 1 mg/ml is optimal for the conjugation reaction, and the lower the concentration, the lower the reaction rate. A wide range of buffers near neutral pH are suitable for coupling/conjugation. The standard choice of conjugation buffer is TBS (20 mM Tris and 150 mM NaCl, pH 7.4). In some cases, Brune et al. Sci Rep. (2016) the addition of a 10x stock of citrate buffer (40 mM Na ₂ HPO ₄ , 200 mM sodium citrate, pH 6.2) can be used.

Pharmaceutical composition and use

The composition of the present invention can be incorporated into a vaccine or immunogenic composition. Suitably, the vaccine or immunogenic composition will contain the particles of the invention in an immunogenic dose.

The pharmaceutical composition may comprise the particles or immunogenic composition according to the present invention provided with a pharmaceutically acceptable carrier. Suitable carriers are well known to those skilled in the art. In one embodiment, the pharmaceutical composition comprises a buffer, excipient or carrier. Suitably, the pharmaceutical composition may contain suitable excipients and agents to maintain the stability of the composition. Suitably, the formulation may comprise an adjuvant. In one embodiment, the agent is a squalene AddaVax ™ or similar having a formulation similar to the MF59 ^® - may comprise an emulsion-based nano-water. Other suitable adjuvants include liposome-based adjuvants such as Matrix M and AS01. Other suitable adjuvants are aluminum, such as Alhydrogel ^® - comprises a base formulation. In one embodiment, the formulation may comprise EDTA at a concentration of 5 mM, for example. Suitable excipients or formulations may vary depending on the properties of the particles or immunogenic composition; For example, the choice of expression system can affect the stability, glycosylation or folding of the protein in the composition, which in turn can affect the optimal formulation of the composition. Methods for determining suitable excipients, agents or adjuvants are known to those skilled in the art.

Various additional aspects and embodiments of the invention will be apparent to those skilled in the art in view of the present disclosure.

All documents mentioned in this specification are incorporated herein by reference in their entirety.

As used herein, “and/or” is to be considered a particular disclosure that includes or does not include each of the two specified features or components. For example, “A and/or B” is the specific disclosure of each of (i) A, (ii) B and (iii) A and B, and should be considered as if each were individually presented herein.

Unless the context indicates otherwise, the descriptions and definitions of the features set forth above are not limited to any particular aspect or embodiment of the invention, and apply equally to all described aspects and embodiments.

Those skilled in the art will also understand that the invention has been described by way of example with reference to several embodiments. This is not limited to the disclosed embodiments, and alternative embodiments may be constructed without departing from the scope of the invention as defined in the appended claims.

As used herein to describe a polynucleotide, “recombinant” refers to a polynucleotide of genomic, cDNA, semisynthetic or synthetic origin, such as, due to its origin or manipulation: (1) essentially bound poly Not associated with all or part of the nucleotides; And/or (2) linked to a polynucleotide other than that which is essentially linked. The term “recombinant” as used in connection with a protein or polypeptide refers to a polypeptide produced by expression of a recombinant polynucleotide.

Unless specifically stated, a method comprising steps can be performed in any suitable order. Thus, the steps can be performed in any suitable order.

Sequence identity between the polypeptide sequences is preferably the Needleman-Wunsch Global, using default parameters (e.g., Gap opening penalty = 10.0 and Gap extension penalty = 0.5, using EBLOSUM62 scoring matrix). It is determined by a pairwise alignment algorithm using an alignment algorithm (Needleman and Wunsch 1970). These algorithms are conveniently implemented with the needle tool from the EMBOSS package (Rice, Longden and Bleasby 2000). Sequence identity should be calculated over the entire length of the polypeptide sequence of the invention.

Any homologues of the components mentioned herein are typically functional homologs, and are typically at least 40% homologous to the relevant region of the protein. Homology can be measured using known methods. For example, the UWGCG package provides a BESTFIT program that can be used to calculate homology (for example, used as its default setting) (Devereux et al (1984) Nucleic Acids Research 12, 387-395). The PILEUP and BLAST algorithms are described, for example, in Altschul S. F. (1993) J Mol Evol 36:290-300; As described in Altschul, S, F et al (1990) J Mol Biol 215:403-10, it can be used to calculate homologous or alignment sequences (typically in their default settings). Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/).

The BLAST algorithm performs statistical analysis of similarity between two sequences; For example, Karlin and Altschul (1993) Proc. Natl. Acad. Sci. See USA 90: 5873-5787. One measure of similarity provided by the BLAST algorithm is the minimum sum probability (P(N)), which represents the probability that a match will occur by chance between two nucleotide or amino acid sequences. For example, if the minimum sum probability in the comparison of the first sequence and the second sequence is less than about 1, preferably less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001, the sequence is a different sequence. Is considered similar to

Variant polypeptides comprise (or consist of) a sequence with at least 40% identity to the native protein. In a preferred embodiment, the variant sequence is at least 20, preferably at least 30, for example at least 40, 60, 100, 200, 300, 400 or more contiguous amino acids for a specific region of the native protein. , Or even at least 55%, 65%, 70%, 75%, 80%, 85%, 90%, more preferably at least 95%, 97% or 99% homology to the entire sequence of the variant. . Alternatively, the variant sequence will be at least 55%, 65%, 70%, 75%, 80%, 85%, 90%, more preferably at least 95%, 97% or 99% homologous to the full-length native protein. I can. Typically the variant sequence can differ from the relevant region of the native protein by at least 2, 5, 10, 20, 40, 50 or less than 60 mutations (each can be a substitution, insertion or deletion). The variant sequence of the invention may have a percent identity with a specific region of the full-length native protein, which is equal to any specific percent homology value over the length of the sequence mentioned above (i.e., it is at least 40%, 55 %, 80% or 90%, more preferably at least 95%, 97% or 99% identity).

Variants of these proteins also include truncations. Any cleavage can be used as long as the variant is still functional. Cleavage will typically be performed to remove sequences that are not essential for activity/function, in particular isopeptide bond formation, and/or do not affect the conformation of the folded protein, particularly folding of any immunogenic site. Cleavage may also be selected to facilitate the preparation of the above components. Proper cleavage can be routinely identified by systematic cleavage of sequences of various lengths from the N- or C-terminus.

The variant of the natural protein is one or more, for example 2, 3, 4, 5 to 10, 10 to 20, 20 to 40 amino acid insertions, substitutions or deletions for a specific region of the natural protein. Include as. Deletion and insertion are preferably carried out outside the antigenic region. Insertion is typically carried out at the N- or C-terminal end of a sequence derived from a native protein, for example for the purpose of recombinant expression. Substitutions are also typically performed in regions that are not essential for activity/function and/or do not affect the conformation of the folded protein. Such substitutions can be made to improve the solubility or other characteristics of the protein. Substitution may be performed to increase the stability of the protein.

The substitution introduces one or more conservative changes, preferably replacing an amino acid with another amino acid of a similar chemical structure, similar chemical property or similar side chain volume. The introduced amino acids may have a polarity, hydrophilicity, hydrophobicity, basicity, acidity, neutrality, or charge similar to the amino acid they replace. As an alternative, the conservative change can introduce other amino acids that are aromatic or aliphatic in place of the existing aromatic or aliphatic amino acids. Conservative amino acid changes are well known in the art.

A derivative is an entity derived or created from a parent entity by replacement of a portion of the parent entity.

Example

Example One

Illustrative Multimer - VLP Composition ( HCMV Pentamer - HBsAg VLP ) Of

The HCMV pentamer was transiently expressed in Expi293F cells using ExpiFectamine™ 293 Transfection Reagent (ThermoFisher Scientific) and five separate plasmids encoding the following sequences. The HCMV pentamer described below is approximately 162 kDa, not glycosylated (including tags and linkers, but excluding signal peptides).

Nucleotide sequence

The expressed HCMV pentameric sequence represents the native sequence from the Merlin strain (GenBank: AY446894.2; low-passage (i.e., attenuated) HCMV strain) (including introns), and the two introduced mutations described in the relevant passages below. (one in gH, one in UL128) is excluded.

gH - SpyTag -His nucleotide sequence (SEQ ID NO: 12)

The sequence (SEQ ID NO: 12), GeneArt ^® synthesis is silent mutations C> A at position 1146 was introduced for, because of such a nucleotide sequence near the natural CACCTGC been shown to have a potential problem. The construct includes: signal peptide (nt 1-69), ectodomain (nt 70-2151), transmembrane domain (cleaved) (nt 2152-2157), (signal peptide, ectodomain and transmembrane domain ( Truncated) together represented by SEQ ID NO: 13), linker (nt 2158-2175; SEQ ID NO: 14), SpyTag (nt 2176-2214; SEQ ID NO: 15), 6x His tag (nt 2215-2232), stop Codon (nt 2233-2235). Nucleotides 1-2157 (SEQ ID NO: 13) represent the gH coding sequence.

gL Nucleotide sequence (SEQ ID NO: 16)

In this sequence: signal peptide (nt 1-90), ectodomain (nt 91-834), stop codon (nt 835-837).

UL130-C-tag nucleotide sequence (SEQ ID NO: 17)

In this sequence: signal peptide (nt 1-75), ectodomain (nt 76-642), linker (nt 643-687), C-tag (nt 688-699), stop codon (nt 700-702).

UL128 nucleotide sequence (SEQ ID NO: 20) (two Intron include)

In this sequence: signal peptide (nt 1-81), intron: nt 165-287, nt 423-542, ectodomain exon (nt 82-164, nt 288-422, nt 543-756), stop codon (nt 757 -759).

The T>C mutation was introduced at nucleotide 634. Since the T634 nucleotide was mentioned in the GenBank file as causing premature termination of UL128 in the Merlin strain, the present inventors tried to find out which base was substituted in returning to the expression of the full-length protein in another strain (GenBank: GQ396662.1, strain HAN38). Was used.

UL131A Nucleotide sequence (SEQ ID NO: 21) (present in the natural sequence Intron include)

In this sequence: signal peptide (nt 1-54), intron (nt 237-344, ectodomain exon (nt 55-236, nt 345-495), stop codon (nt 496-498)).

SpyCatcher - HBsAg Nucleotide sequence (SEQ ID NO: 22)

In this sequence: SpyCatcherDeltaN1 (nt 1-276), flexible linker (nt 277-303), PVTN linker (nt 304-315), HBsAg (nt 316-993), C-tag (nt 994-1005), stop Codon (nt 1006-1008).

Amino acid sequence

Expression of this nucleotide sequence is expected to lead to the following amino acid sequence.

gH - SpyTag -His amino acid sequence (SEQ ID NO: 27)

Expected mass 81.852 kDa (excluding signal peptide), 84.364 kDa (including signal peptide).

In this sequence: signal peptide (aa 1-23), ectodomain (aa 24-717), transmembrane domain (cleaved) (aa 718-719)) (signal peptide, ectodomain and transmembrane domain (cleaved) Together, they are represented by SEQ ID NO: 28), linker (aa 720-725; SEQ ID NO: 29), SpyTag (aa 726-738; SEQ ID NO: 30), 6x His tag (aa 739-744). Amino acid residues 1-719 represent the natural Merlin strain gH amino acid sequence with a truncated TM domain (SEQ ID NO: 28).

gL Amino acid sequence (SEQ ID NO: 31)

Expected mass 27.522 kDa (excluding signal peptide), 30.815 kDa (including signal peptide).

In this sequence: signal peptide (aa 1-30), ectodomain (aa 31-278). Amino acid residues 1-278 represent the natural Merlin strain gL amino acid sequence.

UL130-C-tag amino acid sequence (SEQ ID NO: 32)

Expected mass 23.167 kDa (excluding signal peptide), 26.081 kDa (including signal peptide).

In this sequence: signal peptide (aa 1-25), ectodomain (aa 26-214), (signal peptide and ectodomain together represented by SEQ ID NO: 33), linker (aa 215-229; SEQ ID NO: 34) , C-tag (aa 230-233).

Amino acid residues 1-214 represent the natural Merlin strain UL130 amino acid sequence.

UL128 amino acid sequence (SEQ ID NO: 35)

Expected mass 16.659 kDa (excluding signal peptide), 19.717 kDa (including signal peptide).

In this sequence: signal peptide (aa 1-27), ectodomain (aa 28-171). Amino acid residues 1-171 represent the natural Merlin strain UL128 amino acid sequence.

UL131A Amino acid sequence (SEQ ID NO: 36)

Expected mass 12.985 kDa (excluding signal peptide), 14.989 kDa (including signal peptide).

In the above sequence: signal peptide (aa 1-18), ectodomain (aa 19-129). Amino acid residues 1-129 represent the natural Merlin strain UL131A amino acid sequence.

SpyCatcher - HBsAg Amino acid sequence (SEQ ID NO: 37)

Expected mass including tag and linker 36.824 kDa.

In this sequence: SpyCatcherDeltaN1 (aa 1-92; SEQ ID NO: 38), flexible linker (aa 93-101; SEQ ID NO: 39), PVTN linker (aa 102-105; SEQ ID NO: 40), HBsAg (aa 106 -331; SEQ ID NO: 41), C-tag (aa 332-335).

Pentameric refine

Pentameric-SpyTag was expressed in EXPI293F cells and secreted into the supernatant (due to deletion of the TM domain (part of) from the gH subunit). Early attempts to use affinity purification to purify HCMV pentamers depended on the expression of the C-tagged gH subunit, but this resulted in the isolation of not only pentamers, but also gH/gL heterohomodimers. In an alternative strategy, the supernatant using C-tag affinity purification (ThermoFisher) and size exclusion chromatography by adding a C-tag to the UL130 subunit (SEQ ID NO: 17 (nucleotide) and SEQ ID NO: 32 (amino acid)). The pentamer was purified from The pentamer appeared as expected under non-reducing and reducing conditions when analyzed by SDS-PAGE ( Fig. 1A), and reacted with an anti-HCMV pentameric antibody (Native Antigen Company (AbCMV2450)) ( Fig. 1 B ), minor contaminants were observed at ~14 kDa.

HBsAg VLP Monomer purification

SpyCatcher-HBsAg Pichia Pastoris ( Pichia pastoris ) and purified from cell homogenates. Under the reducing conditions of the SDS-PAGE gel, the dominant protein band corresponds to the expected size of the monomer (approximately 37 kDa), and an additional larger band indicates the presence of an oligomer species, indicating that crosslinking of the particles occurred well. ( A in Fig . 2 , lane'R'). Under non-reducing conditions (lane'NR'), the material mainly remains with some stains on the top of the gel, indicating that the VLP particles are well formed, which is too large to completely migrate to the gel ( FIG. 2A ). Both the non-reduced and reduced SpyCatcher-HBsAg reacted strongly with the mouse anti-HBsAg monoclonal antibody (obtained from Bio-Rad (MCA4658)) ( Fig. 2B ), which indicates that the presence of SpyCatcher negatively affects the reactive epitope. Indicates that

Both HCMV pentamer-SpyTag and SpyCatcher-HBsAg eluted as a single peak as assessed by HPLC size exclusion analysis on the s200increase 3.2/300 column ( AB in FIG. 3 ). HCMV pentameric-SpyTag eluted at ~400 kDa, which is larger than expected ( Fig. 3A ). However, this can be explained by the non-spherical structure of the pentameric known to alter the residence time of the protein during size exclusion chromatography. SpyCatcher-HBsAg eluted in the pore volume of the column, indicating that the particles were properly formed without detectable monomer in solution ( FIG. 3B ).

antigen- VLP join

HCMV pentameric-SpyTag was conjugated to SpyCatcher-HBsAg overnight at 4° C. to generate HBsAg VLPs coated with HCMV-5 mer. A buffer containing Tris buffered saline (TBS; 20 mM Tris and 150 mM NaCl, pH 7.4) supplemented with 5 mM EDTA was used for conjugation. Conjugation was monitored using SDS-PAGE and Western-blot analysis and HPLC. When the conjugation reaction was compared with pentameric-SpyTag or SpyCatcher-HBsAg alone, a new band was present at -130 kDa under reducing conditions ( Fig. 4A , lane 2), which is a monoclonal anti-HBsAg ( Fig. 4 B ) and polyclonal anti-HCMV pentameric ( FIG. 4C ) antibodies reacted with both, indicating that it contains at least the conjugated HBsAg-gH. When analyzed by HPLC size exclusion chromatography, 97% eluted from the main peak corresponding to the expected size of the conjugated HCMV pentameric-HBsAg monomer ( FIG. 5 ).

Example 2

HCMV - SpyTag - SpyCatcher - HBsAg VLP's In vivo test ( Adjuvant use)

Conjugated HCMV pentameric-HBsAg VLP and unconjugated HCMV pentameric-SpyTag were used in the immunization schedule using BALB/c mice, (i) the immunogenicity of the generated HCMV pentameric-SpyTag was confirmed, and (ii) The immunogenicity of unconjugated HCMV pentamer-SpyTag was compared against conjugated HCMV pentamer-HBsAg VLP.

The prime-boost-boost schedule at 3 week intervals was used as follows:

Day 0: Immunization (prime); Day 20: tail blood sampling; Day 21: Immunization (boost 1); Day 41: tail blood sampling; Day 42: Immunization (boost 2); Day 63: Heart blood collection.

The immunized groups are as follows. N = 10 for each group:

1) 1 μg HCMV pentamer-SpyTag in AddaVax™ (Invivogen)

2) 1 μg HCMV pentamer-SpyTag--SpyCatcher-HBsAg VLP in AddaVax™ (1 μg pentameric equivalent)

3) SpyCatcher-HBsAg VLP in AddaVax™ (normalized to the amount of SpyCatcher-HBsAg in Group 2)

4) 0.1 μg HCMV pentamer-SpyTag in AddaVax™

5) 0.1 μg HCMV pentamer-SpyTag--SpyCatcher-HBsAg VLP in AddaVax™ (0.1 μg pentameric equivalent)

6) TBS (20 mM Tris and 150 mM NaCl, pH 7.4)

AddaVax™ is a squalene-based, water-in-water nano-emulsion, similar to MF59 ^® licensed in Europe for adjuvanted flu vaccines. Squalene-in-water emulsions are known to elicit both cellular (Th1) and humoral (Th2) immune responses. Other suitable adjuvants are known to those skilled in the art.

Immunogenicity was evaluated using ELISA. The titer of antisera generated in each group was determined using a standardized ELISA for HCMV pentamer. Plates were coated with 5 μg/ml pentamer (no SpyTag), 50 μL/well overnight; Washing; Blocked with milk for 1 hour; Washing; Mouse serum (suitably diluted in PBS) was applied for 1 hour; Washing; Goat anti-mouse-alkali phosphatase antibody (1:10,000) was applied for 1 hour; Washing; Occurred.

Immunization between the conjugated HCMV pentamer-HBsAg VLP vaccine and the unconjugated HCMV pentamer-SpyTag, including different doses of unconjugated (groups 1 and 4) and conjugated HCMV pentamer-HBsAg (groups 2 and 5) Allows for origin comparison, which was extrapolated to other HCMV pentameric vaccines (eg soluble pentameric). Groups 3 and 6 represent negative controls.

At each time point, the OD values for the samples were read at the appropriate dilution and ELISA units were determined using standard curves run on each plate. After prime, data showing results for groups 1, 2, 4 and 5 are shown in FIG. 6. HCMV pentameric-HBsAg immunized mice using both 1 μg and 0.1 μg doses exhibited substantially stronger serum IgG antibody responses compared to mice immunized with unconjugated HCMV pentamers at 1 μg or 0.1 μg doses. Show. The ELISA units for groups 3 and 6 provided a baseline for this assay and are also shown in Figure 6.

The functional activity of the resulting antibody was determined by Wang et al . (Vaccine 33 (2015) 7254-7261; DOI: 10.1016/j.vaccine. 2015.10.110) based on microneutralisation assay was used to study. Neutralizing titers for groups 1, 2, 4 and 5 are shown in Figure 7 . Serum from mice immunized with pentameric-HBsAg VLPs is substantially more neutralized than serum from mice immunized with pentameric-SpyTag protein alone.

Example 3

Stable construct sequence

Two stable constructs (adapted from Hofmann et al., (2015) Biotech and Bioeng, 112(12):2505-2515) were optimized for CHO expression of the HCMV pentameric-SpyTag component. The intron was removed from the HCMV pentameric sequence, but the signal sequence was retained.

HCMV gH - SpyTag / gL Stable expression construct

Stable vector construct HCMV-gH-(GSG) ₂ -SpyTag-His-IRES- to include the gH-SpyTag-His component (SEQ ID NO: 42) and gL component (SEQ ID NO: 43), respectively, upstream and downstream of the EV71 IRES. The gL was designed. The coding sequence used in the construct is described below.

Nucleotide order

Inserted upstream of the EV71 IRES gH -( GSG )2- SpyTag -His ( Intron None) (SEQ ID NO: 42)

In this sequence: signal peptide (nt 1-69), ectodomain (nt 70-2151), truncated transmembrane domain (nt 2152-2157), (GSG) ₂ linker (nt 2158-2175), SpyTag (nt 2176 -2214), His-tag (nt 2215-2232), stop codon (nt 2233-2235).

Inserted downstream of the EV71 IRES gL ( Intron None) (SEQ ID NO: 43)

In this sequence: signal peptide (nt 1-90), ectodomain (nt 91-834), stop codon (nt 835-837)

HCMV UL128/UL130/ UL131A Stable expression construct

Stable construct HCMV-UL128-IRES-UL130-(G4S)3-C-tag-IRES- to include UL128 component (SEQ ID NO: 44), UL130 component (SEQ ID NO: 45) and UL131A component (SEQ ID NO: 46) UL131A was designed. The UL130 component was inserted after the first EV71 IRES of the plasmid, and the UL131A component was inserted after the second EV71 IRES. The coding sequences used in this construct are described below.

Nucleotide sequence

UL128 ( Intron None) (SEQ ID NO: 44)

In this sequence: signal peptide (nt 1-81), ectodomain (nt 82-513), stop codon (nt 514-516).

UL130-( G4S )3-C-tag ( Intron None) (SEQ ID NO: 45)

In this sequence: signal peptide (nt 1-75), ectodomain (nt 76-642), (G4S) ₃ linker (nt 643-687), C tag (nt 688-699), stop codon (nt 700-702 ).

UL131A ( Intron None) (SEQ ID NO: 46)

In this sequence: signal peptide (nt 1-54), ectodomain (nt 55-387), stop codon (nt 388-390).

Example 4

HCMV - SpyTag - SpyCatcher - HBsAg VLP's In vivo test ( Adjuvant none)

In order to further study the immunogenicity of the conjugated HCMV pentameric-HBsAg VLP and the unconjugated HCMV pentameric-SpyTag, the conjugated pentameric-HBsAg VLP against the unconjugated pentameric-SpyTag protein, BALB/c Used in the immunization schedule with mice.

The prime-boost-boost schedule at 3 week intervals was used as follows:

Day 0: Immunization (prime); Day 20: tail blood sampling; Day 21: Immunization (boost 1); Day 41: tail blood sampling; Day 42: Immunization (boost 2); Day 63: Heart blood collection.

The immunized groups are as follows. N = 10 for each group:

1) 1 μg HCMV pentamer-SpyTag no adjuvant

2) 1 μg HCMV pentameric-SpyTag--SpyCatcher-HBsAg VLP (1 μg pentameric equivalent) no adjuvant

3) 0.1 μg HCMV pentameric-SpyTag--SpyCatcher-HBsAg VLP (0.1 μg pentameric equivalent) no adjuvant

Immunogenicity was evaluated using ELISA. The titer of antisera generated in each group was determined using a standardized ELISA for HCMV pentamer. Plates were coated with 5 μg/ml pentamer (no SpyTag), 50 μL/well overnight; Washing; Blocked with milk for 1 hour; Washing; Mouse serum (suitably diluted in PBS) was applied for 1 hour; Washing; Goat anti-mouse-alkali phosphatase antibody (1:10,000) was applied for 1 hour; Washing; Occurred.

At each time point, the OD values for the samples were read at the appropriate dilution and the ELISA unit was determined using a standard curve run on each plate. Post-prime and post-boost data are shown in FIG. 8 . HCMV pentameric-HBsAg immunized mice using both 1 μg and 0.1 μg doses show a substantially stronger serum IgG antibody response compared to mice immunized with 1 μg of HCMV pentamer alone as soluble protein.

The functional activity of the resulting antibody was determined by Wang et al . (2015) based on microneutralization analysis was used to study. Neutralization titers after prime and after boost are shown in FIG. 9 . Serum from mice immunized with adjuvant-free pentameric-HBsAg VLPs is substantially more neutralized than serum from mice immunized with adjuvant-free pentameric-SpyTag protein alone.

Example 5

RSV -F- SpyTag Expression and purification

The sequence from antigen RSV-F Sc9-10 DS-Cav1 A149C Y458C was fused to SpyTag to generate RSV-F-SpyTag, and using ExpiCHO™ expression system kit and ExpiFectamine™ transfection reagent (ThermoFisher Scientific), plasmid pcDNA3 ExpiCHO™ cells were transiently transfected and expressed with the nucleotide sequence SEQ ID NO: 47 in .4.

RSV-F Sc9-10 DS-Cav1 A149C Y458C (National Institutes of Health), Joyce et al. (2016), it is a variant of a respiratory syncytial virus fusion glycoprotein vaccine.Nat Struct Mol Biol. 2016 Sep; 23(Iterative structure-based improvement of a respiratory syncytial virus fusion glycoprotein vaccine. 9): 811-820). These variants include gene-linked F subunits, deleted fusion peptides, T4 fibritin trimerization motifs (foldon domains) and interprotomer movements stabilized by additional interprotomeric disulfide bonds (A149C Y458C). Fusion (F) is a pre-fusion stabilized form of a glycoprotein.

Nucleotide sequence

RSV-F-SpyTag-C tag nucleotide sequence (SEQ ID NO: 47)

The original sequence of Sc9-10 DS-Cav1 A149C Y458C was modified at the 3'end by deletion of the thrombin site, 6x His-tag and Strep-tag ^® II. By replacing these deleted domains with a linker-SpyTag-C-tag sequence, Sc9-10 DS-Cav1 A149C Y458C (nt 1-1515, signal peptide (nt 1-75), and T4 fibritin foldon domain (nt 1435) -1515)), (GSG) ₂ linker (nt 1516-1533; SEQ ID NO: 14), SpyTag (nt 1534-1572; SEQ ID NO: 15), C-tag (nt 1573-1584) and stop codon (nt 1587 nt cassette (SEQ ID NO: 47)) containing 1585-1587) was created. The Sc9-10 DS-Cav1 A149C Y458C nucleotide sequence excluding linker, SpyTag, C-tag and stop codon is included in SEQ ID NO: 48. The Sc9-10 DS-Cav1 A149C Y458C nucleotide acid sequence excluding the signal peptide, linker, SpyTag or C-tag is included in SEQ ID NO: 49.

Amino acid sequence

Expression of the nucleotide sequence SEQ ID NO: 47 was expected to produce an RSV-F-SpyTag-C tag amino acid sequence (SEQ ID NO: 50) having the following domain: Sc9-10 DS-Cav1 A149C Y458C ((aa 1-505 , Including signal peptide (aa 1-25) and foldon domain (aa 479-505)), linker (aa 506-511; SEQ ID NO: 29), SpyTag (aa 512-524; SEQ ID NO: 30), C-tag (aa 525-528) The expected mass of the protein was 57.9 kDa with signal peptide and 55.3 kDa without signal peptide Sc9-10 DS-Cav1 A149C Y458C amino acid sequence excluding linker, SpyTag or C-tag It is included in SEQ ID NO: 51. The Sc9-10 DS-Cav1 A149C Y458C amino acid sequence excluding signal peptide, linker, SpyTag or C-tag is included in SEQ ID NO: 52.

RSV -F- SpyTag refine

RSF-F-SpyTag antigen was secreted from the cells and purified from the supernatant using C-tag affinity purification and size exclusion chromatography. RSV-F-SpyTag appeared as expected under non-reducing and reducing conditions when analyzed by SDS-PAGE ( Fig. 10A ), and reacted with anti-RSV-F [2F7] monoclonal antibody (ab43812; Abcam) ( Fig. 10B ).

HBsAg VLP Monomer purification

SpyCatcher-HBsAg (VLP monomer) was prepared and purified as described in Example 1 above, see also FIG. 2.

RSV -F- SpyTag SpyCatcher - To HBsAg join

RSV-F-SpyTag was conjugated to SpyCatcher-HBsAg at 4° C. overnight to generate HBsAg VLPs coated with RSV-F trimer (RSV-F-SpyTag--SpyCatcher-HBsAg). A buffer containing Tris buffered saline (TBS; 20 mM Tris and 150 mM NaCl, pH 7.4) was used for conjugation. The conjugation was monitored using SDS-PAGE and Western-blot analysis ( FIG. 11 ). When the conjugation reaction was compared with RSV-F-SpyTag or SpyCatcher-HBsAg alone, a new band was present at ~105 kDa (lane 2) under reducing conditions ( Fig. 11A ), which is an anti-HBsAg monoclonal antibody. (MCA4658, Bio-Rad) ( FIG. 11B ) and anti-RSV-F [2F7] monoclonal antibody (ab43812; Abcam) ( FIG. 11C ) reacted with both, which is conjugated RSV-F-SpyTag - Indicates that it contains SpyCatcher-HBsAg.

Example 6

Spliced RSV -F- SpyTag - SpyCatcher - HBsAg Immunogenicity

To confirm the immunogenicity of the generated RSV-F antigen and to compare the immunogenicity of the conjugated RSV-F-SpyTag--SpyCatcher-HBsAg VLP to the unconjugated RSV-F-SpyTag protein, BALB/c mice were Was used to design an immunization schedule. The groups were administered based on the amount of RSV-F-SpyTag in the sample, and a prime-boost schedule of 3-week intervals was selected, with a final time point 2 weeks after boost immunization.

After prime immunization with RSV-F-SpyTag--SpyCatcher-HBsAg, mice, regardless of whether the vaccine did not use adjuvant ( FIG. 6 ) or formulated with Addavax™ ( FIG. 6 ), RSV-F- Compared to mice immunized with SpyTag protein alone, it showed a substantially stronger serum IgG antibody response.

SEQUENCE LISTING <110> SpyBiotech Limited <120> Vaccine composition <130> P9800wo1 <160> 60 <170> PatentIn version 3.5 <210> 1 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Flexible linker 2-(GSG)2 <400> 1 Gly Ser Gly Gly Ser Gly 1 5 <210> 2 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Flexible linker 3-(GSG)3 <400> 2 Gly Ser Gly Gly Ser Gly Gly Ser Gly 1 5 <210> 3 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Flexible linker 4-(G4S)1 <400> 3 Gly Gly Gly Gly Ser 1 5 <210> 4 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Flexible linker 5-(G4S)3 <400> 4 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 <210> 5 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Flexible linker 6-(G4S)4 <400> 5 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser 20 <210> 6 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Flexible linker 7 <400> 6 Gly Ser Ala Gly Ser Ala Ala Gly Ser Gly Glu Phe 1 5 10 <210> 7 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Flexible linker 8 <400> 7 Lys Glu Ser Gly Ser Val Ser Ser Glu Gln Leu Ala Gln Phe Arg Ser 1 5 10 15 Leu Asp <210> 8 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Flexible linker 9 <400> 8 Glu Gly Lys Ser Ser Gly Ser Gly Ser Glu Ser Lys Ser Thr 1 5 10 <210> 9 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Rigid linker 1 <400> 9 Glu Ala Ala Ala Lys 1 5 <210> 10 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Rigid linker 2-(EAAAK)3 <400> 10 Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys 1 5 10 15 <210> 11 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Rigid linker 3-(AP)7 <400> 11 Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro 1 5 10 <210> 12 <211> 2235 <212> DNA <213> Artificial Sequence <220> <223> gH-SpyTag-His <220> <221> misc_feature <222> (1146)..(1146) <223> C>A mutation at position 1146 <220> <221> misc_feature <222> (1)..(69) <223> Signal peptide <220> <221> misc_feature <222> (70)..(2151) <223> Ectodomain <220> <221> misc_feature <222> (2152)..(2157) <223> Transmembrane domain (truncated) <220> <221> misc_feature <222> (2158)..(2175) <223> Linker <220> <221> misc_feature <222> (2176)..(2214) <223> SpyTag <220> <221> misc_feature <222> (2215)..(2232) <223> 6x His tag <220> <221> misc_feature <222> (2233)..(2235) <223> Stop codon <400> 12 atgcggccag gcctcccctc ctacctcatc atcctcgccg tctgtctctt cagccaccta 60 ctttcgtcac gatatggcgc agaagccgta tccgaaccgc tggacaaagc gtttcaccta 120 ctgctcaaca cctacgggag acccatccgc ttcctgcgtg aaaataccac ccagtgtacc 180 tacaacagca gcctccgtaa cagcacggtc gtcagggaaa acgccatcag tttcaacttt 240 ttccaaagct ataatcaata ctatgtattc catatgcctc gatgtctttt tgcgggtcct 300 ctggcggagc agtttctgaa ccaggtagat ctgaccgaaa ccctggaaag ataccaacag 360 agacttaaca cttacgcgct ggtatccaaa gacctggcca gctaccgatc tttttcgcag 420 cagctaaagg cacaagacag cctaggtgaa cagcccacca ctgtgccacc gcccattgac 480 ctgtcaatac ctcacgtttg gatgccaccg caaaccactc cacacggctg gacagaatca 540 cataccacct caggactaca ccgaccacac tttaaccaga cctgtatcct ctttgatgga 600 cacgatctac tattcagcac cgtcacacct tgtttgcacc aaggctttta cctcatcgac 660 gaactacgtt acgttaaaat aacactgacc gaggacttct tcgtagttac ggtgtccata 720 gacgacgaca cacccatgct gcttatcttc ggccatcttc cacgcgtact tttcaaagcg 780 ccctatcaac gcgacaactt tatactacga caaactgaaa aacacgagct cctggtgcta 840 gttaagaaag atcaactgaa ccgtcactct tatctcaaag acccggactt tcttgacgcc 900 gcacttgact tcaactacct agacctcagc gcactactac gtaacagctt tcaccgttac 960 gccgtggatg tactcaagag cggtcgatgt cagatgctgg accgccgcac ggtagaaatg 1020 gccttcgcct acgcattagc actgttcgca gcagcccgac aagaagaggc cggcgcccaa 1080 gtctccgtcc cacgggccct agaccgccag gccgcactct tacaaataca agaatttatg 1140 atcacatgcc tctcacaaac accaccacgc accacgttgc tgctgtatcc cacggccgtg 1200 gacctggcca aacgagccct ttggacaccg aatcagatca ccgacatcac cagcctcgta 1260 cgcctggtct acatactctc taaacagaat cagcaacatc tcatccccca atgggcacta 1320 cgacagatcg ccgactttgc cctaaaacta cacaaaacgc acctggcctc ttttctttca 1380 gccttcgcac gccaagaact ctacctcatg ggcagcctcg tccactccat gctggtacat 1440 acgacggaga gacgcgaaat cttcatcgta gaaacgggcc tctgttcatt ggccgagcta 1500 tcacacttta cgcagttgtt agctcatcca caccacgaat acctcagcga cctgtacaca 1560 ccctgttcca gtagcgggcg acgcgatcac tcgctcgaac gcctcacgcg tctcttcccc 1620 gatgccaccg tccccgctac cgttcccgcc gccctctcca tcctatctac catgcaacca 1680 agcacgctgg aaaccttccc cgacctgttt tgcttgccgc tcggcgaatc cttctccgcg 1740 ctgaccgtct ccgaacacgt cagttatatc gtaacaaacc agtacctgat caaaggtatc 1800 tcctaccctg tctccaccac cgtcgtaggc cagagcctca tcatcaccca gacggacagt 1860 caaactaaat gcgaactgac gcgcaacatg cataccacac acagcatcac agtggcgctc 1920 aacatttcgc tagaaaactg cgccttttgc caaagcgccc tgctagaata cgacgacacg 1980 caaggcgtca tcaacatcat gtacatgcac gactcggacg acgtcctttt cgccctggat 2040 ccctacaacg aagtggtggt ctcatctccg cgaactcact acctcatgct tttgaaaaac 2100 ggtacggtac tagaagtaac tgacgtcgtc gtggacgcca ccgacagtcg tctcctcgga 2160 agcggaggct ctggtgccca tatcgtgatg gtggacgcct acaagcctac caaacatcat 2220 caccatcacc actaa 2235 <210> 13 <211> 2157 <212> DNA <213> Artificial Sequence <220> <223> gH with truncated transmembrane domain <220> <221> misc_feature <222> (1146)..(1146) <223> C>A mutation at position 1146 <220> <221> misc_feature <222> (1)..(69) <223> Signal peptide <220> <221> misc_feature <222> (70)..(2151) <223> Ectodomain <220> <221> misc_feature <222> (2152)..(2157) <223> Transmembrane domain (truncated) <400> 13 atgcggccag gcctcccctc ctacctcatc atcctcgccg tctgtctctt cagccaccta 60 ctttcgtcac gatatggcgc agaagccgta tccgaaccgc tggacaaagc gtttcaccta 120 ctgctcaaca cctacgggag acccatccgc ttcctgcgtg aaaataccac ccagtgtacc 180 tacaacagca gcctccgtaa cagcacggtc gtcagggaaa acgccatcag tttcaacttt 240 ttccaaagct ataatcaata ctatgtattc catatgcctc gatgtctttt tgcgggtcct 300 ctggcggagc agtttctgaa ccaggtagat ctgaccgaaa ccctggaaag ataccaacag 360 agacttaaca cttacgcgct ggtatccaaa gacctggcca gctaccgatc tttttcgcag 420 cagctaaagg cacaagacag cctaggtgaa cagcccacca ctgtgccacc gcccattgac 480 ctgtcaatac ctcacgtttg gatgccaccg caaaccactc cacacggctg gacagaatca 540 cataccacct caggactaca ccgaccacac tttaaccaga cctgtatcct ctttgatgga 600 cacgatctac tattcagcac cgtcacacct tgtttgcacc aaggctttta cctcatcgac 660 gaactacgtt acgttaaaat aacactgacc gaggacttct tcgtagttac ggtgtccata 720 gacgacgaca cacccatgct gcttatcttc ggccatcttc cacgcgtact tttcaaagcg 780 ccctatcaac gcgacaactt tatactacga caaactgaaa aacacgagct cctggtgcta 840 gttaagaaag atcaactgaa ccgtcactct tatctcaaag acccggactt tcttgacgcc 900 gcacttgact tcaactacct agacctcagc gcactactac gtaacagctt tcaccgttac 960 gccgtggatg tactcaagag cggtcgatgt cagatgctgg accgccgcac ggtagaaatg 1020 gccttcgcct acgcattagc actgttcgca gcagcccgac aagaagaggc cggcgcccaa 1080 gtctccgtcc cacgggccct agaccgccag gccgcactct tacaaataca agaatttatg 1140 atcacatgcc tctcacaaac accaccacgc accacgttgc tgctgtatcc cacggccgtg 1200 gacctggcca aacgagccct ttggacaccg aatcagatca ccgacatcac cagcctcgta 1260 cgcctggtct acatactctc taaacagaat cagcaacatc tcatccccca atgggcacta 1320 cgacagatcg ccgactttgc cctaaaacta cacaaaacgc acctggcctc ttttctttca 1380 gccttcgcac gccaagaact ctacctcatg ggcagcctcg tccactccat gctggtacat 1440 acgacggaga gacgcgaaat cttcatcgta gaaacgggcc tctgttcatt ggccgagcta 1500 tcacacttta cgcagttgtt agctcatcca caccacgaat acctcagcga cctgtacaca 1560 ccctgttcca gtagcgggcg acgcgatcac tcgctcgaac gcctcacgcg tctcttcccc 1620 gatgccaccg tccccgctac cgttcccgcc gccctctcca tcctatctac catgcaacca 1680 agcacgctgg aaaccttccc cgacctgttt tgcttgccgc tcggcgaatc cttctccgcg 1740 ctgaccgtct ccgaacacgt cagttatatc gtaacaaacc agtacctgat caaaggtatc 1800 tcctaccctg tctccaccac cgtcgtaggc cagagcctca tcatcaccca gacggacagt 1860 caaactaaat gcgaactgac gcgcaacatg cataccacac acagcatcac agtggcgctc 1920 aacatttcgc tagaaaactg cgccttttgc caaagcgccc tgctagaata cgacgacacg 1980 caaggcgtca tcaacatcat gtacatgcac gactcggacg acgtcctttt cgccctggat 2040 ccctacaacg aagtggtggt ctcatctccg cgaactcact acctcatgct tttgaaaaac 2100 ggtacggtac tagaagtaac tgacgtcgtc gtggacgcca ccgacagtcg tctcctc 2157 <210> 14 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Linker from gH construct <400> 14 ggaagcggag gctctggt 18 <210> 15 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Spytag <400> 15 gcccatatcg tgatggtgga cgcctacaag cctaccaaa 39 <210> 16 <211> 837 <212> DNA <213> Human cytomegalovirus <220> <221> misc_feature <222> (1)..(90) <223> Signal peptide <220> <221> misc_feature <222> (91)..(834) <223> Ectodomain <220> <221> misc_feature <222> (835)..(837) <223> Stop codon <400> 16 atgtgccgcc gcccggattg cggcttctct ttctcacctg gaccggtgat actgctgtgg 60 tgttgccttc tgctgcccat tgtttcctca gccgccgtca gcgtcgctcc taccgccgcc 120 gagaaagtcc ccgcggagtg ccccgaacta acgcgccgat gcttgttggg tgaggtgttt 180 gagggtgaca agtatgaaag ttggctgcgc ccgttggtga atgttaccgg gcgcgatggc 240 ccgctatcgc aacttatccg ttaccgtccc gttacgccgg aggccgccaa ctccgtgctg 300 ttggacgagg ctttcctgga cactctggcc ctgctgtaca acaatccgga tcaattgcgg 360 gccctgctga cgctgttgag ctcggacaca gcgccgcgct ggatgacggt gatgcgcggc 420 tacagcgagt gcggcgatgg ctcgccggcc gtgtacacgt gcgtggacga cctgtgccgc 480 ggctacgacc tcacgcgact gtcatacggg cgcagcatct tcacggaaca cgtgttaggc 540 ttcgagctgg tgccaccgtc tctctttaac gtggtggtgg ccatacgcaa cgaagccacg 600 cgtaccaacc gcgccgtgcg tctgcccgtg agcaccgctg ccgcgcccga gggcatcacg 660 ctcttttacg gcctgtacaa cgcagtgaag gaattctgcc tgcgtcacca gctggacccg 720 ccgctgctac gccacctaga taaatactac gccggactgc cgcccgagct gaagcagacg 780 cgcgtcaacc tgccggctca ctcgcgctat ggccctcaag cagtggatgc tcgctaa 837 <210> 17 <211> 702 <212> DNA <213> Artificial Sequence <220> <223> UL130-C-tag <220> <221> misc_feature <222> (1)..(75) <223> Signal peptide <220> <221> misc_feature <222> (76)..(642) <223> Ectodomain <220> <221> misc_feature <222> (643)..(687) <223> Linker <220> <221> misc_feature <222> (688)..(699) <223> C-tag <220> <221> misc_feature <222> (700)..(702) <223> Stop codon <400> 17 atgctgcggc ttctgcttcg tcaccacttt cactgcctgc ttctgtgcgc ggtttgggca 60 acgccctgtc tggcgtctcc gtggtcgacg ctaacagcaa accagaatcc gtccccgcca 120 tggtctaaac tgacgtattc caaaccgcat gacgcggcga cgttttactg tccttttctc 180 tatccctcgc ccccacgatc ccccttgcaa ttctcggggt tccagcgggt atcaacgggt 240 cccgagtgtc gcaacgagac cctgtatctg ctgtacaacc gggaaggcca gaccttggtg 300 gagagaagct ccacctgggt gaaaaaggtg atctggtacc tgagcggtcg gaaccaaacc 360 atcctccaac ggatgccccg aacggcttcg aaaccgagcg acggaaacgt gcagatcagc 420 gtggaagacg ccaagatttt tggagcgcac atggtgccca agcagaccaa gctgctacgc 480 ttcgtcgtca acgatggcac acgttatcag atgtgtgtga tgaagctgga gagctgggct 540 cacgtcttcc gggactacag cgtgtctttt caggtgcgat tgacgttcac cgaggccaat 600 aaccagactt acaccttctg cacccatccc aatctcatcg ttggaggcgg aggatctggc 660 ggaggtggaa gtggcggagg cggatctgag cccgaggcct aa 702 <210> 18 <211> 642 <212> DNA <213> Artificial Sequence <220> <223> UL130 (signal sequence and ectodomain) <220> <221> misc_feature <222> (1)..(75) <223> Signal peptide <220> <221> misc_feature <222> (76)..(642) <223> Ectodomain <400> 18 atgctgcggc ttctgcttcg tcaccacttt cactgcctgc ttctgtgcgc ggtttgggca 60 acgccctgtc tggcgtctcc gtggtcgacg ctaacagcaa accagaatcc gtccccgcca 120 tggtctaaac tgacgtattc caaaccgcat gacgcggcga cgttttactg tccttttctc 180 tatccctcgc ccccacgatc ccccttgcaa ttctcggggt tccagcgggt atcaacgggt 240 cccgagtgtc gcaacgagac cctgtatctg ctgtacaacc gggaaggcca gaccttggtg 300 gagagaagct ccacctgggt gaaaaaggtg atctggtacc tgagcggtcg gaaccaaacc 360 atcctccaac ggatgccccg aacggcttcg aaaccgagcg acggaaacgt gcagatcagc 420 gtggaagacg ccaagatttt tggagcgcac atggtgccca agcagaccaa gctgctacgc 480 ttcgtcgtca acgatggcac acgttatcag atgtgtgtga tgaagctgga gagctgggct 540 cacgtcttcc gggactacag cgtgtctttt caggtgcgat tgacgttcac cgaggccaat 600 aaccagactt acaccttctg cacccatccc aatctcatcg tt 642 <210> 19 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Linker from UL130 construct <400> 19 ggaggcggag gatctggcgg aggtggaagt ggcggaggcg gatct 45 <210> 20 <211> 759 <212> DNA <213> Human cytomegalovirus <220> <221> misc_feature <222> (634)..(634) <223> T>C mutation at position 634 <220> <221> misc_feature <222> (1)..(81) <223> Signal peptide <220> <221> misc_feature <222> (82)..(164) <223> Ectodomain <220> <221> Intron <222> (165)..(287) <220> <221> misc_feature <222> (288)..(422) <223> Ectodomain <220> <221> Intron <222> (423)..(542) <220> <221> misc_feature <222> (543)..(756) <223> Ectodomain <220> <221> misc_feature <222> (757)..(759) <223> Stop codon <400> 20 atgagtccca aagatctgac gccgttcttg acggcgttgt ggctgctatt gggtcacagc 60 cgcgtgccgc gggtgcgcgc agaagaatgt tgcgaattca taaacgtcaa ccacccgccg 120 gaacgctgtt acgatttcaa aatgtgcaat cgcttcaccg tcgcgtacgt attttcatga 180 ttgtctgcgt tctgtggtgc gtctggatct gtctctcgac gtttctgata gccatgttcc 240 atcgacgatc ctcgggaatg ccagagtaga ttttcatgaa tccacaggct gcggtgtccg 300 gacggcgaag tctgctacag tcccgagaaa acggctgaga ttcgcgggat cgtcaccacc 360 atgacccatt cattgacacg ccaggtcgta cacaacaaac tgacgagctg caactacaat 420 ccgtaagtct cttcctgagg gccttacagc ctatgggaga gtaagacaga gagggacaaa 480 acatcattaa aaaaaaaagt ctaatttcac gttttgtacc ccccttcccc tccgtgttgt 540 aggttatacc tcgaagctga cgggcgaata cgctgcggca aagtaaacga caaggcgcag 600 tacctgctgg gcgccgctgg cagcgttccc tatcgatgga tcaatctgga atacgacaag 660 ataacccgga tcgtgggcct ggatcagtac ctggagagcg ttaagaaaca caaacggctg 720 gatgtgtgcc gcgctaaaat gggctatatg ctgcagtga 759 <210> 21 <211> 498 <212> DNA <213> Human cytomegalovirus <220> <221> misc_feature <222> (1)..(54) <223> Signal peptide <220> <221> misc_feature <222> (55)..(236) <223> Ectodomain <220> <221> Intron <222> (237)..(344) <220> <221> misc_feature <222> (345)..(495) <223> Ectodomain <220> <221> misc_feature <222> (496)..(498) <223> Stop codon <400> 21 atgcggctgt gtcgggtgtg gctgtctgtt tgtctgtgcg ccgtggtgct gggtcagtgc 60 cagcgggaaa ccgcggaaaa aaacgattat taccgagtac cgcattactg ggacgcgtgc 120 tctcgcgcgc tgcccgacca aacccgttac aagtatgtgg aacagctcgt ggacctcacg 180 ttgaactacc actacgatgc gagccacggc ttggacaact ttgacgtgct caagaggtga 240 gggtacgcgc taaagatgca tgacaacggg aaggtaaggg cgaacgggta acgggtaagt 300 aaccgcatgg ggtatgaaat gacgttcgga acctgtgctt gcagaatcaa cgtgaccgag 360 gtgtcgttgc tcatcagcga ctttagacgt cagaaccgtc gcggcggcac caacaaaagg 420 accacgttca acgccgccgg ttcgctggcg ccacacgccc ggagcctcga gttcagcgtg 480 cggctctttg ccaactag 498 <210> 22 <211> 1008 <212> DNA <213> Artificial Sequence <220> <223> SpyCatcher-HBsAg <220> <221> misc_feature <222> (1)..(276) <223> SpyCatcherDeltaN1 <220> <221> misc_feature <222> (277)..(303) <223> Flexible Linker <220> <221> misc_feature <222> (304)..(315) <223> PVTN linker <220> <221> misc_feature <222> (316)..(993) <223> HBsAg <220> <221> misc_feature <222> (994)..(1005) <223> C-tag <220> <221> misc_feature <222> (1006)..(1008) <223> Stop codon <400> 22 gactccgcta ctcacatcaa gttctccaag agagatgagg acggtaaaga attggctggt 60 gctactatgg aattgagaga ctcctccggt aagactatct ccacttggat ttccgacggt 120 caggttaagg acttctactt gtacccaggt aagtacactt tcgttgagac tgctgctcca 180 gacggttacg aagttgctac tgctatcact ttcactgtta acgagcaggg acaggttaca 240 gttaacggta aggctactaa gggtgacgct catattggtt ctggtggatc tggtggttcc 300 ggtccagtta ctaatatgga aaacatcact tccggtttct tgggtccttt gttggttttg 360 caggctggat tcttcttgtt gactagaatc ttgactatcc cacagtcctt ggactcttgg 420 tggacttcct tgaacttctt gggtggttcc ccagtttgtt tgggtcaaaa ctctcaatcc 480 ccaacttcca accactcccc aacatcttgt ccaccaattt gtcctggtta cagatggatg 540 tgtttgagaa gattcatcat tttcttgttc atcttgttgt tgtgtttgat cttcttgttg 600 gttttgttgg actaccaggg tatgttgcca gtttgtccat tgatcccagg ttccactact 660 acaaacactg gtccatgtaa gacttgtact actccagctc agggtaactc catgttccct 720 tcatgttgtt gtactaagcc aactgacggt aactgtactt gtatcccaat tccatcctcc 780 tgggctttcg ctaagtactt gtgggaatgg gcttccgtta gattctcctg gttgtccttg 840 ttggttccat tcgttcagtg gttcgttggt ttgtccccaa ctgtttggtt gtccgctatt 900 tggatgatgt ggtactgggg tccatccttg tactctatcg tttccccatt catccctttg 960 ttgccaatct tcttctgttt gtgggtttac atcgagccag aggcttaa 1008 <210> 23 <211> 276 <212> DNA <213> Artificial Sequence <220> <223> SpyCatcherDeltaN1 <400> 23 gactccgcta ctcacatcaa gttctccaag agagatgagg acggtaaaga attggctggt 60 gctactatgg aattgagaga ctcctccggt aagactatct ccacttggat ttccgacggt 120 caggttaagg acttctactt gtacccaggt aagtacactt tcgttgagac tgctgctcca 180 gacggttacg aagttgctac tgctatcact ttcactgtta acgagcaggg acaggttaca 240 gttaacggta aggctactaa gggtgacgct catatt 276 <210> 24 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Flexible linker from SpyCatcher-HBsAg <400> 24 ggttctggtg gatctggtgg ttccggt 27 <210> 25 <211> 12 <212> DNA <213> Artificial Sequence <220> <223> PVTN linker from SpyCatcher-HbsAg <400> 25 ccagttacta at 12 <210> 26 <211> 678 <212> DNA <213> Artificial Sequence <220> <223> HBsAg <400> 26 atggaaaaca tcacttccgg tttcttgggt cctttgttgg ttttgcaggc tggattcttc 60 ttgttgacta gaatcttgac tatcccacag tccttggact cttggtggac ttccttgaac 120 ttcttgggtg gttccccagt ttgtttgggt caaaactctc aatccccaac ttccaaccac 180 tccccaacat cttgtccacc aatttgtcct ggttacagat ggatgtgttt gagaagattc 240 atcattttct tgttcatctt gttgttgtgt ttgatcttct tgttggtttt gttggactac 300 cagggtatgt tgccagtttg tccattgatc ccaggttcca ctactacaaa cactggtcca 360 tgtaagactt gtactactcc agctcagggt aactccatgt tcccttcatg ttgttgtact 420 aagccaactg acggtaactg tacttgtatc ccaattccat cctcctgggc tttcgctaag 480 tacttgtggg aatgggcttc cgttagattc tcctggttgt ccttgttggt tccattcgtt 540 cagtggttcg ttggtttgtc cccaactgtt tggttgtccg ctatttggat gatgtggtac 600 tggggtccat ccttgtactc tatcgtttcc ccattcatcc ctttgttgcc aatcttcttc 660 tgtttgtggg tttacatc 678 <210> 27 <211> 744 <212> PRT <213> Artificial Sequence <220> <223> gH-SpyTag-His <220> <221> misc_feature <222> (1)..(23) <223> Signal peptide <220> <221> misc_feature <222> (24)..(717) <223> Ectodomain <220> <221> misc_feature <222> (718)..(719) <223> Transmembrane domain (truncated) <220> <221> misc_feature <222> (720)..(725) <223> Linker <220> <221> misc_feature <222> (726)..(738) <223> Spytag <220> <221> misc_feature <222> (739)..(744) <223> 6x His tag <400> 27 Met Arg Pro Gly Leu Pro Ser Tyr Leu Ile Ile Leu Ala Val Cys Leu 1 5 10 15 Phe Ser His Leu Leu Ser Ser Arg Tyr Gly Ala Glu Ala Val Ser Glu 20 25 30 Pro Leu Asp Lys Ala Phe His Leu Leu Leu Asn Thr Tyr Gly Arg Pro 35 40 45 Ile Arg Phe Leu Arg Glu Asn Thr Thr Gln Cys Thr Tyr Asn Ser Ser 50 55 60 Leu Arg Asn Ser Thr Val Val Arg Glu Asn Ala Ile Ser Phe Asn Phe 65 70 75 80 Phe Gln Ser Tyr Asn Gln Tyr Tyr Val Phe His Met Pro Arg Cys Leu 85 90 95 Phe Ala Gly Pro Leu Ala Glu Gln Phe Leu Asn Gln Val Asp Leu Thr 100 105 110 Glu Thr Leu Glu Arg Tyr Gln Gln Arg Leu Asn Thr Tyr Ala Leu Val 115 120 125 Ser Lys Asp Leu Ala Ser Tyr Arg Ser Phe Ser Gln Gln Leu Lys Ala 130 135 140 Gln Asp Ser Leu Gly Glu Gln Pro Thr Thr Val Pro Pro Pro Ile Asp 145 150 155 160 Leu Ser Ile Pro His Val Trp Met Pro Pro Gln Thr Thr Pro His Gly 165 170 175 Trp Thr Glu Ser His Thr Thr Ser Gly Leu His Arg Pro His Phe Asn 180 185 190 Gln Thr Cys Ile Leu Phe Asp Gly His Asp Leu Leu Phe Ser Thr Val 195 200 205 Thr Pro Cys Leu His Gln Gly Phe Tyr Leu Ile Asp Glu Leu Arg Tyr 210 215 220 Val Lys Ile Thr Leu Thr Glu Asp Phe Phe Val Val Thr Val Ser Ile 225 230 235 240 Asp Asp Asp Thr Pro Met Leu Leu Ile Phe Gly His Leu Pro Arg Val 245 250 255 Leu Phe Lys Ala Pro Tyr Gln Arg Asp Asn Phe Ile Leu Arg Gln Thr 260 265 270 Glu Lys His Glu Leu Leu Val Leu Val Lys Lys Asp Gln Leu Asn Arg 275 280 285 His Ser Tyr Leu Lys Asp Pro Asp Phe Leu Asp Ala Ala Leu Asp Phe 290 295 300 Asn Tyr Leu Asp Leu Ser Ala Leu Leu Arg Asn Ser Phe His Arg Tyr 305 310 315 320 Ala Val Asp Val Leu Lys Ser Gly Arg Cys Gln Met Leu Asp Arg Arg 325 330 335 Thr Val Glu Met Ala Phe Ala Tyr Ala Leu Ala Leu Phe Ala Ala Ala 340 345 350 Arg Gln Glu Glu Ala Gly Ala Gln Val Ser Val Pro Arg Ala Leu Asp 355 360 365 Arg Gln Ala Ala Leu Leu Gln Ile Gln Glu Phe Met Ile Thr Cys Leu 370 375 380 Ser Gln Thr Pro Pro Arg Thr Thr Leu Leu Leu Tyr Pro Thr Ala Val 385 390 395 400 Asp Leu Ala Lys Arg Ala Leu Trp Thr Pro Asn Gln Ile Thr Asp Ile 405 410 415 Thr Ser Leu Val Arg Leu Val Tyr Ile Leu Ser Lys Gln Asn Gln Gln 420 425 430 His Leu Ile Pro Gln Trp Ala Leu Arg Gln Ile Ala Asp Phe Ala Leu 435 440 445 Lys Leu His Lys Thr His Leu Ala Ser Phe Leu Ser Ala Phe Ala Arg 450 455 460 Gln Glu Leu Tyr Leu Met Gly Ser Leu Val His Ser Met Leu Val His 465 470 475 480 Thr Thr Glu Arg Arg Glu Ile Phe Ile Val Glu Thr Gly Leu Cys Ser 485 490 495 Leu Ala Glu Leu Ser His Phe Thr Gln Leu Leu Ala His Pro His His 500 505 510 Glu Tyr Leu Ser Asp Leu Tyr Thr Pro Cys Ser Ser Ser Gly Arg Arg 515 520 525 Asp His Ser Leu Glu Arg Leu Thr Arg Leu Phe Pro Asp Ala Thr Val 530 535 540 Pro Ala Thr Val Pro Ala Ala Leu Ser Ile Leu Ser Thr Met Gln Pro 545 550 555 560 Ser Thr Leu Glu Thr Phe Pro Asp Leu Phe Cys Leu Pro Leu Gly Glu 565 570 575 Ser Phe Ser Ala Leu Thr Val Ser Glu His Val Ser Tyr Ile Val Thr 580 585 590 Asn Gln Tyr Leu Ile Lys Gly Ile Ser Tyr Pro Val Ser Thr Thr Val 595 600 605 Val Gly Gln Ser Leu Ile Ile Thr Gln Thr Asp Ser Gln Thr Lys Cys 610 615 620 Glu Leu Thr Arg Asn Met His Thr Thr His Ser Ile Thr Val Ala Leu 625 630 635 640 Asn Ile Ser Leu Glu Asn Cys Ala Phe Cys Gln Ser Ala Leu Leu Glu 645 650 655 Tyr Asp Asp Thr Gln Gly Val Ile Asn Ile Met Tyr Met His Asp Ser 660 665 670 Asp Asp Val Leu Phe Ala Leu Asp Pro Tyr Asn Glu Val Val Val Ser 675 680 685 Ser Pro Arg Thr His Tyr Leu Met Leu Leu Lys Asn Gly Thr Val Leu 690 695 700 Glu Val Thr Asp Val Val Val Asp Ala Thr Asp Ser Arg Leu Leu Gly 705 710 715 720 Ser Gly Gly Ser Gly Ala His Ile Val Met Val Asp Ala Tyr Lys Pro 725 730 735 Thr Lys His His His His His His 740 <210> 28 <211> 719 <212> PRT <213> Artificial Sequence <220> <223> gH with truncated transmembrane domain <220> <221> misc_feature <222> (1)..(23) <223> Signal peptide <220> <221> misc_feature <222> (24)..(717) <223> Ectodomain <220> <221> misc_feature <222> (718)..(719) <223> Transmembrane domain (truncated) <400> 28 Met Arg Pro Gly Leu Pro Ser Tyr Leu Ile Ile Leu Ala Val Cys Leu 1 5 10 15 Phe Ser His Leu Leu Ser Ser Arg Tyr Gly Ala Glu Ala Val Ser Glu 20 25 30 Pro Leu Asp Lys Ala Phe His Leu Leu Leu Asn Thr Tyr Gly Arg Pro 35 40 45 Ile Arg Phe Leu Arg Glu Asn Thr Thr Gln Cys Thr Tyr Asn Ser Ser 50 55 60 Leu Arg Asn Ser Thr Val Val Arg Glu Asn Ala Ile Ser Phe Asn Phe 65 70 75 80 Phe Gln Ser Tyr Asn Gln Tyr Tyr Val Phe His Met Pro Arg Cys Leu 85 90 95 Phe Ala Gly Pro Leu Ala Glu Gln Phe Leu Asn Gln Val Asp Leu Thr 100 105 110 Glu Thr Leu Glu Arg Tyr Gln Gln Arg Leu Asn Thr Tyr Ala Leu Val 115 120 125 Ser Lys Asp Leu Ala Ser Tyr Arg Ser Phe Ser Gln Gln Leu Lys Ala 130 135 140 Gln Asp Ser Leu Gly Glu Gln Pro Thr Thr Val Pro Pro Pro Ile Asp 145 150 155 160 Leu Ser Ile Pro His Val Trp Met Pro Pro Gln Thr Thr Pro His Gly 165 170 175 Trp Thr Glu Ser His Thr Thr Ser Gly Leu His Arg Pro His Phe Asn 180 185 190 Gln Thr Cys Ile Leu Phe Asp Gly His Asp Leu Leu Phe Ser Thr Val 195 200 205 Thr Pro Cys Leu His Gln Gly Phe Tyr Leu Ile Asp Glu Leu Arg Tyr 210 215 220 Val Lys Ile Thr Leu Thr Glu Asp Phe Phe Val Val Thr Val Ser Ile 225 230 235 240 Asp Asp Asp Thr Pro Met Leu Leu Ile Phe Gly His Leu Pro Arg Val 245 250 255 Leu Phe Lys Ala Pro Tyr Gln Arg Asp Asn Phe Ile Leu Arg Gln Thr 260 265 270 Glu Lys His Glu Leu Leu Val Leu Val Lys Lys Asp Gln Leu Asn Arg 275 280 285 His Ser Tyr Leu Lys Asp Pro Asp Phe Leu Asp Ala Ala Leu Asp Phe 290 295 300 Asn Tyr Leu Asp Leu Ser Ala Leu Leu Arg Asn Ser Phe His Arg Tyr 305 310 315 320 Ala Val Asp Val Leu Lys Ser Gly Arg Cys Gln Met Leu Asp Arg Arg 325 330 335 Thr Val Glu Met Ala Phe Ala Tyr Ala Leu Ala Leu Phe Ala Ala Ala 340 345 350 Arg Gln Glu Glu Ala Gly Ala Gln Val Ser Val Pro Arg Ala Leu Asp 355 360 365 Arg Gln Ala Ala Leu Leu Gln Ile Gln Glu Phe Met Ile Thr Cys Leu 370 375 380 Ser Gln Thr Pro Pro Arg Thr Thr Leu Leu Leu Tyr Pro Thr Ala Val 385 390 395 400 Asp Leu Ala Lys Arg Ala Leu Trp Thr Pro Asn Gln Ile Thr Asp Ile 405 410 415 Thr Ser Leu Val Arg Leu Val Tyr Ile Leu Ser Lys Gln Asn Gln Gln 420 425 430 His Leu Ile Pro Gln Trp Ala Leu Arg Gln Ile Ala Asp Phe Ala Leu 435 440 445 Lys Leu His Lys Thr His Leu Ala Ser Phe Leu Ser Ala Phe Ala Arg 450 455 460 Gln Glu Leu Tyr Leu Met Gly Ser Leu Val His Ser Met Leu Val His 465 470 475 480 Thr Thr Glu Arg Arg Glu Ile Phe Ile Val Glu Thr Gly Leu Cys Ser 485 490 495 Leu Ala Glu Leu Ser His Phe Thr Gln Leu Leu Ala His Pro His His 500 505 510 Glu Tyr Leu Ser Asp Leu Tyr Thr Pro Cys Ser Ser Ser Gly Arg Arg 515 520 525 Asp His Ser Leu Glu Arg Leu Thr Arg Leu Phe Pro Asp Ala Thr Val 530 535 540 Pro Ala Thr Val Pro Ala Ala Leu Ser Ile Leu Ser Thr Met Gln Pro 545 550 555 560 Ser Thr Leu Glu Thr Phe Pro Asp Leu Phe Cys Leu Pro Leu Gly Glu 565 570 575 Ser Phe Ser Ala Leu Thr Val Ser Glu His Val Ser Tyr Ile Val Thr 580 585 590 Asn Gln Tyr Leu Ile Lys Gly Ile Ser Tyr Pro Val Ser Thr Thr Val 595 600 605 Val Gly Gln Ser Leu Ile Ile Thr Gln Thr Asp Ser Gln Thr Lys Cys 610 615 620 Glu Leu Thr Arg Asn Met His Thr Thr His Ser Ile Thr Val Ala Leu 625 630 635 640 Asn Ile Ser Leu Glu Asn Cys Ala Phe Cys Gln Ser Ala Leu Leu Glu 645 650 655 Tyr Asp Asp Thr Gln Gly Val Ile Asn Ile Met Tyr Met His Asp Ser 660 665 670 Asp Asp Val Leu Phe Ala Leu Asp Pro Tyr Asn Glu Val Val Val Ser 675 680 685 Ser Pro Arg Thr His Tyr Leu Met Leu Leu Lys Asn Gly Thr Val Leu 690 695 700 Glu Val Thr Asp Val Val Val Asp Ala Thr Asp Ser Arg Leu Leu 705 710 715 <210> 29 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Linker from gH construct <400> 29 Gly Ser Gly Gly Ser Gly 1 5 <210> 30 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Spytag <400> 30 Ala His Ile Val Met Val Asp Ala Tyr Lys Pro Thr Lys 1 5 10 <210> 31 <211> 278 <212> PRT <213> Human cytomegalovirus <220> <221> misc_feature <222> (1)..(30) <223> Signal peptide <220> <221> misc_feature <222> (31)..(278) <223> Ectodomain <400> 31 Met Cys Arg Arg Pro Asp Cys Gly Phe Ser Phe Ser Pro Gly Pro Val 1 5 10 15 Ile Leu Leu Trp Cys Cys Leu Leu Leu Pro Ile Val Ser Ser Ala Ala 20 25 30 Val Ser Val Ala Pro Thr Ala Ala Glu Lys Val Pro Ala Glu Cys Pro 35 40 45 Glu Leu Thr Arg Arg Cys Leu Leu Gly Glu Val Phe Glu Gly Asp Lys 50 55 60 Tyr Glu Ser Trp Leu Arg Pro Leu Val Asn Val Thr Gly Arg Asp Gly 65 70 75 80 Pro Leu Ser Gln Leu Ile Arg Tyr Arg Pro Val Thr Pro Glu Ala Ala 85 90 95 Asn Ser Val Leu Leu Asp Glu Ala Phe Leu Asp Thr Leu Ala Leu Leu 100 105 110 Tyr Asn Asn Pro Asp Gln Leu Arg Ala Leu Leu Thr Leu Leu Ser Ser 115 120 125 Asp Thr Ala Pro Arg Trp Met Thr Val Met Arg Gly Tyr Ser Glu Cys 130 135 140 Gly Asp Gly Ser Pro Ala Val Tyr Thr Cys Val Asp Asp Leu Cys Arg 145 150 155 160 Gly Tyr Asp Leu Thr Arg Leu Ser Tyr Gly Arg Ser Ile Phe Thr Glu 165 170 175 His Val Leu Gly Phe Glu Leu Val Pro Pro Ser Leu Phe Asn Val Val 180 185 190 Val Ala Ile Arg Asn Glu Ala Thr Arg Thr Asn Arg Ala Val Arg Leu 195 200 205 Pro Val Ser Thr Ala Ala Ala Pro Glu Gly Ile Thr Leu Phe Tyr Gly 210 215 220 Leu Tyr Asn Ala Val Lys Glu Phe Cys Leu Arg His Gln Leu Asp Pro 225 230 235 240 Pro Leu Leu Arg His Leu Asp Lys Tyr Tyr Ala Gly Leu Pro Pro Glu 245 250 255 Leu Lys Gln Thr Arg Val Asn Leu Pro Ala His Ser Arg Tyr Gly Pro 260 265 270 Gln Ala Val Asp Ala Arg 275 <210> 32 <211> 233 <212> PRT <213> Artificial Sequence <220> <223> UL130-C-tag <220> <221> misc_feature <222> (1)..(25) <223> Signal peptide <220> <221> misc_feature <222> (26)..(214) <223> Ectodomain <220> <221> misc_feature <222> (215)..(229) <223> Linker <220> <221> misc_feature <222> (230)..(233) <223> C-tag <400> 32 Met Leu Arg Leu Leu Leu Arg His His Phe His Cys Leu Leu Leu Cys 1 5 10 15 Ala Val Trp Ala Thr Pro Cys Leu Ala Ser Pro Trp Ser Thr Leu Thr 20 25 30 Ala Asn Gln Asn Pro Ser Pro Pro Trp Ser Lys Leu Thr Tyr Ser Lys 35 40 45 Pro His Asp Ala Ala Thr Phe Tyr Cys Pro Phe Leu Tyr Pro Ser Pro 50 55 60 Pro Arg Ser Pro Leu Gln Phe Ser Gly Phe Gln Arg Val Ser Thr Gly 65 70 75 80 Pro Glu Cys Arg Asn Glu Thr Leu Tyr Leu Leu Tyr Asn Arg Glu Gly 85 90 95 Gln Thr Leu Val Glu Arg Ser Ser Thr Trp Val Lys Lys Val Ile Trp 100 105 110 Tyr Leu Ser Gly Arg Asn Gln Thr Ile Leu Gln Arg Met Pro Arg Thr 115 120 125 Ala Ser Lys Pro Ser Asp Gly Asn Val Gln Ile Ser Val Glu Asp Ala 130 135 140 Lys Ile Phe Gly Ala His Met Val Pro Lys Gln Thr Lys Leu Leu Arg 145 150 155 160 Phe Val Val Asn Asp Gly Thr Arg Tyr Gln Met Cys Val Met Lys Leu 165 170 175 Glu Ser Trp Ala His Val Phe Arg Asp Tyr Ser Val Ser Phe Gln Val 180 185 190 Arg Leu Thr Phe Thr Glu Ala Asn Asn Gln Thr Tyr Thr Phe Cys Thr 195 200 205 His Pro Asn Leu Ile Val Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 210 215 220 Gly Gly Gly Gly Ser Glu Pro Glu Ala 225 230 <210> 33 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> UL130 (signal sequence and ectodomain) <220> <221> misc_feature <222> (1)..(25) <223> Signal peptide <220> <221> misc_feature <222> (26)..(214) <223> Ectodomain <400> 33 Met Leu Arg Leu Leu Leu Arg His His Phe His Cys Leu Leu Leu Cys 1 5 10 15 Ala Val Trp Ala Thr Pro Cys Leu Ala Ser Pro Trp Ser Thr Leu Thr 20 25 30 Ala Asn Gln Asn Pro Ser Pro Pro Trp Ser Lys Leu Thr Tyr Ser Lys 35 40 45 Pro His Asp Ala Ala Thr Phe Tyr Cys Pro Phe Leu Tyr Pro Ser Pro 50 55 60 Pro Arg Ser Pro Leu Gln Phe Ser Gly Phe Gln Arg Val Ser Thr Gly 65 70 75 80 Pro Glu Cys Arg Asn Glu Thr Leu Tyr Leu Leu Tyr Asn Arg Glu Gly 85 90 95 Gln Thr Leu Val Glu Arg Ser Ser Thr Trp Val Lys Lys Val Ile Trp 100 105 110 Tyr Leu Ser Gly Arg Asn Gln Thr Ile Leu Gln Arg Met Pro Arg Thr 115 120 125 Ala Ser Lys Pro Ser Asp Gly Asn Val Gln Ile Ser Val Glu Asp Ala 130 135 140 Lys Ile Phe Gly Ala His Met Val Pro Lys Gln Thr Lys Leu Leu Arg 145 150 155 160 Phe Val Val Asn Asp Gly Thr Arg Tyr Gln Met Cys Val Met Lys Leu 165 170 175 Glu Ser Trp Ala His Val Phe Arg Asp Tyr Ser Val Ser Phe Gln Val 180 185 190 Arg Leu Thr Phe Thr Glu Ala Asn Asn Gln Thr Tyr Thr Phe Cys Thr 195 200 205 His Pro Asn Leu Ile Val 210 <210> 34 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Linker from UL130 construct <400> 34 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 <210> 35 <211> 171 <212> PRT <213> Human cytomegalovirus <220> <221> misc_feature <222> (1)..(27) <223> Signal peptide <220> <221> misc_feature <222> (28)..(171) <223> Ectodomain <400> 35 Met Ser Pro Lys Asp Leu Thr Pro Phe Leu Thr Ala Leu Trp Leu Leu 1 5 10 15 Leu Gly His Ser Arg Val Pro Arg Val Arg Ala Glu Glu Cys Cys Glu 20 25 30 Phe Ile Asn Val Asn His Pro Pro Glu Arg Cys Tyr Asp Phe Lys Met 35 40 45 Cys Asn Arg Phe Thr Val Ala Leu Arg Cys Pro Asp Gly Glu Val Cys 50 55 60 Tyr Ser Pro Glu Lys Thr Ala Glu Ile Arg Gly Ile Val Thr Thr Met 65 70 75 80 Thr His Ser Leu Thr Arg Gln Val Val His Asn Lys Leu Thr Ser Cys 85 90 95 Asn Tyr Asn Pro Leu Tyr Leu Glu Ala Asp Gly Arg Ile Arg Cys Gly 100 105 110 Lys Val Asn Asp Lys Ala Gln Tyr Leu Leu Gly Ala Ala Gly Ser Val 115 120 125 Pro Tyr Arg Trp Ile Asn Leu Glu Tyr Asp Lys Ile Thr Arg Ile Val 130 135 140 Gly Leu Asp Gln Tyr Leu Glu Ser Val Lys Lys His Lys Arg Leu Asp 145 150 155 160 Val Cys Arg Ala Lys Met Gly Tyr Met Leu Gln 165 170 <210> 36 <211> 129 <212> PRT <213> Human cytomegalovirus <220> <221> misc_feature <222> (1)..(18) <223> Signal peptide <220> <221> misc_feature <222> (19)..(129) <223> Ectodomain <400> 36 Met Arg Leu Cys Arg Val Trp Leu Ser Val Cys Leu Cys Ala Val Val 1 5 10 15 Leu Gly Gln Cys Gln Arg Glu Thr Ala Glu Lys Asn Asp Tyr Tyr Arg 20 25 30 Val Pro His Tyr Trp Asp Ala Cys Ser Arg Ala Leu Pro Asp Gln Thr 35 40 45 Arg Tyr Lys Tyr Val Glu Gln Leu Val Asp Leu Thr Leu Asn Tyr His 50 55 60 Tyr Asp Ala Ser His Gly Leu Asp Asn Phe Asp Val Leu Lys Arg Ile 65 70 75 80 Asn Val Thr Glu Val Ser Leu Leu Ile Ser Asp Phe Arg Arg Gln Asn 85 90 95 Arg Arg Gly Gly Thr Asn Lys Arg Thr Thr Phe Asn Ala Ala Gly Ser 100 105 110 Leu Ala Pro His Ala Arg Ser Leu Glu Phe Ser Val Arg Leu Phe Ala 115 120 125 Asn <210> 37 <211> 335 <212> PRT <213> Artificial Sequence <220> <223> SpyCatcher-HBsAg <220> <221> misc_feature <222> (1)..(92) <223> SpycatcherDeltaN1 <220> <221> misc_feature <222> (93)..(101) <223> Flexible Linker <220> <221> misc_feature <222> (102)..(105) <223> PVTN linker <220> <221> misc_feature <222> (106)..(331) <223> HBsAg <220> <221> misc_feature <222> (332)..(335) <223> C-tag <400> 37 Asp Ser Ala Thr His Ile Lys Phe Ser Lys Arg Asp Glu Asp Gly Lys 1 5 10 15 Glu Leu Ala Gly Ala Thr Met Glu Leu Arg Asp Ser Ser Gly Lys Thr 20 25 30 Ile Ser Thr Trp Ile Ser Asp Gly Gln Val Lys Asp Phe Tyr Leu Tyr 35 40 45 Pro Gly Lys Tyr Thr Phe Val Glu Thr Ala Ala Pro Asp Gly Tyr Glu 50 55 60 Val Ala Thr Ala Ile Thr Phe Thr Val Asn Glu Gln Gly Gln Val Thr 65 70 75 80 Val Asn Gly Lys Ala Thr Lys Gly Asp Ala His Ile Gly Ser Gly Gly 85 90 95 Ser Gly Gly Ser Gly Pro Val Thr Asn Met Glu Asn Ile Thr Ser Gly 100 105 110 Phe Leu Gly Pro Leu Leu Val Leu Gln Ala Gly Phe Phe Leu Leu Thr 115 120 125 Arg Ile Leu Thr Ile Pro Gln Ser Leu Asp Ser Trp Trp Thr Ser Leu 130 135 140 Asn Phe Leu Gly Gly Ser Pro Val Cys Leu Gly Gln Asn Ser Gln Ser 145 150 155 160 Pro Thr Ser Asn His Ser Pro Thr Ser Cys Pro Pro Ile Cys Pro Gly 165 170 175 Tyr Arg Trp Met Cys Leu Arg Arg Phe Ile Ile Phe Leu Phe Ile Leu 180 185 190 Leu Leu Cys Leu Ile Phe Leu Leu Val Leu Leu Asp Tyr Gln Gly Met 195 200 205 Leu Pro Val Cys Pro Leu Ile Pro Gly Ser Thr Thr Thr Asn Thr Gly 210 215 220 Pro Cys Lys Thr Cys Thr Thr Pro Ala Gln Gly Asn Ser Met Phe Pro 225 230 235 240 Ser Cys Cys Cys Thr Lys Pro Thr Asp Gly Asn Cys Thr Cys Ile Pro 245 250 255 Ile Pro Ser Ser Trp Ala Phe Ala Lys Tyr Leu Trp Glu Trp Ala Ser 260 265 270 Val Arg Phe Ser Trp Leu Ser Leu Leu Val Pro Phe Val Gln Trp Phe 275 280 285 Val Gly Leu Ser Pro Thr Val Trp Leu Ser Ala Ile Trp Met Met Trp 290 295 300 Tyr Trp Gly Pro Ser Leu Tyr Ser Ile Val Ser Pro Phe Ile Pro Leu 305 310 315 320 Leu Pro Ile Phe Phe Cys Leu Trp Val Tyr Ile Glu Pro Glu Ala 325 330 335 <210> 38 <211> 92 <212> PRT <213> Artificial Sequence <220> <223> SpyCatcherDeltaN1 <400> 38 Asp Ser Ala Thr His Ile Lys Phe Ser Lys Arg Asp Glu Asp Gly Lys 1 5 10 15 Glu Leu Ala Gly Ala Thr Met Glu Leu Arg Asp Ser Ser Gly Lys Thr 20 25 30 Ile Ser Thr Trp Ile Ser Asp Gly Gln Val Lys Asp Phe Tyr Leu Tyr 35 40 45 Pro Gly Lys Tyr Thr Phe Val Glu Thr Ala Ala Pro Asp Gly Tyr Glu 50 55 60 Val Ala Thr Ala Ile Thr Phe Thr Val Asn Glu Gln Gly Gln Val Thr 65 70 75 80 Val Asn Gly Lys Ala Thr Lys Gly Asp Ala His Ile 85 90 <210> 39 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Flexible linker from SpyCatcher-HBsAg <400> 39 Gly Ser Gly Gly Ser Gly Gly Ser Gly 1 5 <210> 40 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> PVTN linker from SpyCatcher-HBsAg <400> 40 Pro Val Thr Asn One <210> 41 <211> 226 <212> PRT <213> Artificial Sequence <220> <223> HBsAg <400> 41 Met Glu Asn Ile Thr Ser Gly Phe Leu Gly Pro Leu Leu Val Leu Gln 1 5 10 15 Ala Gly Phe Phe Leu Leu Thr Arg Ile Leu Thr Ile Pro Gln Ser Leu 20 25 30 Asp Ser Trp Trp Thr Ser Leu Asn Phe Leu Gly Gly Ser Pro Val Cys 35 40 45 Leu Gly Gln Asn Ser Gln Ser Pro Thr Ser Asn His Ser Pro Thr Ser 50 55 60 Cys Pro Pro Ile Cys Pro Gly Tyr Arg Trp Met Cys Leu Arg Arg Phe 65 70 75 80 Ile Ile Phe Leu Phe Ile Leu Leu Leu Cys Leu Ile Phe Leu Leu Val 85 90 95 Leu Leu Asp Tyr Gln Gly Met Leu Pro Val Cys Pro Leu Ile Pro Gly 100 105 110 Ser Thr Thr Thr Asn Thr Gly Pro Cys Lys Thr Cys Thr Thr Pro Ala 115 120 125 Gln Gly Asn Ser Met Phe Pro Ser Cys Cys Cys Thr Lys Pro Thr Asp 130 135 140 Gly Asn Cys Thr Cys Ile Pro Ile Pro Ser Ser Trp Ala Phe Ala Lys 145 150 155 160 Tyr Leu Trp Glu Trp Ala Ser Val Arg Phe Ser Trp Leu Ser Leu Leu 165 170 175 Val Pro Phe Val Gln Trp Phe Val Gly Leu Ser Pro Thr Val Trp Leu 180 185 190 Ser Ala Ile Trp Met Met Trp Tyr Trp Gly Pro Ser Leu Tyr Ser Ile 195 200 205 Val Ser Pro Phe Ile Pro Leu Leu Pro Ile Phe Phe Cys Leu Trp Val 210 215 220 Tyr Ile 225 <210> 42 <211> 2235 <212> DNA <213> Artificial Sequence <220> <223> gH-(GSG)2-SpyTag-His (without introns) optimized for CHO expression <220> <221> misc_feature <222> (1)..(69) <223> gH signal peptide <220> <221> misc_feature <222> (70)..(2151) <223> gH ectodomain <220> <221> misc_feature <222> (2152)..(2157) <223> gH truncated transmembrane domain <220> <221> misc_feature <222> (2158)..(2175) <223> (GSG)2 linker <220> <221> misc_feature <222> (2176)..(2214) <223> Spytag <220> <221> misc_feature <222> (2215)..(2232) <223> Histag <220> <221> misc_feature <222> (2233)..(2235) <223> Stop codon <400> 42 atgaggcctg gcctgccttc ttatctgatc atcctggccg tgtgcctgtt ctcccatctg 60 ctgtcctcta gatacggcgc cgaggctgtg tctgagcctc tggataaggc ctttcatctg 120 ctgctgaaca cctacggcag acctatccgg ttcctgcgcg agaacaccac acagtgcacc 180 tacaactcca gcctgcggaa ctccacagtc gtgcgggaaa acgccatctc cttcaacttt 240 ttccagtcct acaaccagta ctatgtgttc cacatgcctc gctgcctgtt cgctggacct 300 ctggctgagc agttcctgaa ccaggtggac ctgaccgaga cactggaaag ataccagcag 360 cggctgaaca catatgccct ggtgtctaag gacctggcct cctacagatc cttcagccag 420 cagctgaagg ctcaggactc tctgggagag cagcctacaa cagtgcctcc tcctatcgac 480 ctgtctatcc ctcacgtgtg gatgcctcca cagaccacac ctcatggctg gaccgagtct 540 cataccacct ctggcctgca ccggcctcac ttcaaccaga cctgcatcct gttcgacggc 600 cacgacctgc tgttctccac cgtgacacca tgtctgcacc agggcttcta cctgatcgac 660 gagctgagat acgtgaagat taccctgaca gaggacttct tcgtggtcac cgtgtccatc 720 gacgacgaca cccctatgct gctgatcttc ggccatctgc ctcgggtgct gttcaaggcc 780 ccttaccagc gggacaactt catcctgaga cagaccgaga agcacgagct gctggtgctg 840 gtcaagaagg accagctgaa ccggcactcc tacctgaagg accctgactt cctggacgcc 900 gctctggact tcaactacct ggatctgagc gccctgctgc ggaacagctt tcacagatac 960 gccgtggacg tgctgaagtc tggcagatgc cagatgctgg acagacggac cgtggaaatg 1020 gccttcgctt acgccctggc tctgtttgcc gccgctagac aagaagaggc tggcgcccaa 1080 gtgtccgtgc ctagagcact ggatagacaa gccgctctgc tgcagatcca agagttcatg 1140 atcacatgcc tgtctcagac ccctcctcgg accacactgc tgctgtatcc taccgctgtg 1200 gatctggcca agagggctct gtggacccct aaccagatca ccgacatcac atccctcgtg 1260 cggctggtgt acatcctgtc caagcagaac cagcagcatc tgatccctca gtgggccctg 1320 aggcagatcg ctgattttgc cctgaagctg cacaagaccc acctggccag ctttctgtct 1380 gccttcgcca gacaagagct gtacctgatg ggcagcctgg tgcactctat gctggtgcat 1440 accaccgagc ggcgcgagat cttcatcgtg gaaaccggcc tgtgttccct ggccgagctg 1500 tctcacttta cccagctgct cgctcaccct caccacgagt acctgtccga cctgtacacc 1560 ccttgctcct ctagcggcag aagggaccac agcctggaaa gactgacccg gctgttccct 1620 gatgccaccg tgcctgctac agttcctgcc gctctgtcca tcctgagcac catgcagcct 1680 tccactctgg aaacattccc cgacctgttc tgcctgcctc tgggcgagtc tttttctgcc 1740 ctgaccgtgt ccgagcacgt gtcctacatc gtgaccaatc agtacctgat caagggcatc 1800 agctaccccg tgtccacaac cgtcgttggc cagagcctga tcatcaccca gaccgactct 1860 cagaccaagt gcgagctgac ccggaacatg cacacaaccc actccatcac cgtggctctg 1920 aacatctccc tggaaaactg cgccttctgc cagtctgccc tgctggaata cgatgacacc 1980 cagggcgtga tcaacatcat gtatatgcac gactccgacg acgtgctgtt tgccctggat 2040 ccttacaacg aggtggtggt gtctagcccc agaacacact acctgatgct gctcaagaac 2100 ggcaccgtgc tggaagtgac cgacgtggtg gtggacgcca ccgattctag attgctcggc 2160 tctggtggct ccggcgctca tatcgtgatg gtggatgctt acaagcccac caagcaccat 2220 catcaccacc actaa 2235 <210> 43 <211> 837 <212> DNA <213> Artificial Sequence <220> <223> gL (without introns) optimized for CHO expression <220> <221> misc_feature <222> (1)..(90) <223> gL signal peptide <220> <221> misc_feature <222> (91)..(834) <223> gL ectodomain <220> <221> misc_feature <222> (835)..(837) <223> Stop codon <400> 43 atgtgcagaa ggcctgactg cggcttctcc ttctctcccg gacctgtgat cctgctgtgg 60 tgctgtctgc tgctgcccat cgtttcttcc gccgctgtgt ctgtggctcc taccgctgct 120 gaaaaggtgc cagctgagtg tcccgagctg accagaagat gtctgctggg cgaagtgttc 180 gagggcgata agtacgagtc ttggctgcgg cctctggtca acgtgaccgg aagagatgga 240 cccctgagcc agctgatccg gtacagacct gtgacacctg aggccgccaa ttccgtgctg 300 ctggatgagg ccttcctgga cacactggcc ctgctgtaca acaaccccga tcagctgaga 360 gccctgctga ccctgctgtc ctctgatacc gctcctagat ggatgaccgt gatgcggggc 420 tactctgagt gcggagatgg aagcccagcc gtgtacacct gtgtggacga tctgtgcaga 480 ggctacgacc tgaccagact gtcctacggc cggtccatct ttaccgagca tgtgctgggc 540 tttgagctgg tgcctcctag cctgttcaat gtggtggtgg ccatccggaa tgaggccacc 600 agaacaaata gagccgtgcg gctgcctgtg tctacagctg ctgctcctga gggcatcacc 660 ctgttctacg gcctgtacaa cgccgtgaaa gagttctgcc tgagacacca gctggaccct 720 ccactgctga ggcacctgga taagtactac gctggcctgc ctcctgagct gaagcagacc 780 agagtgaacc tgcctgctca ctccagatac ggccctcagg ctgtggacgc cagataa 837 <210> 44 <211> 516 <212> DNA <213> Artificial Sequence <220> <223> UL128 (without introns) optimized for CHO expression <220> <221> misc_feature <222> (1)..(81) <223> UL128 signal peptide <220> <221> misc_feature <222> (82)..(513) <223> UL128 ectodomain <220> <221> misc_feature <222> (514)..(516) <223> Stop codon <400> 44 atgtccccta aggatctgac ccctttcctg accgctctgt ggctgctgct gggccattct 60 agagtgccta gagtcagagc cgaggaatgc tgcgagttca tcaacgtgaa ccatcctcca 120 gagcggtgct acgacttcaa gatgtgcaac agattcaccg tggctctgcg gtgccctgat 180 ggcgaagtgt gctactcccc tgaaaagacc gccgagatca gaggcatcgt gaccaccatg 240 acacactccc tgaccagaca ggtggtgcac aacaagctga ccagctgcaa ctacaaccct 300 ctgtacctgg aagccgacgg cagaatcaga tgcggcaaag tgaacgacaa ggcccagtac 360 ctgttgggcg ctgctggctc tgtgccctac agatggatca acctggaata cgacaagatc 420 acccggatcg tcggcctgga ccagtatctg gaatccgtga agaagcacaa gcggctggac 480 gtgtgcagag ccaagatggg ctatatgctg cagtaa 516 <210> 45 <211> 702 <212> DNA <213> Artificial Sequence <220> <223> UL130-(G4S)3-C-tag (without introns) optimized for CHO expression <220> <221> misc_feature <222> (1)..(75) <223> UL130 signal peptide <220> <221> misc_feature <222> (76)..(642) <223> UL130 ectodomain <220> <221> misc_feature <222> (643)..(687) <223> (G4S)3 linker <220> <221> misc_feature <222> (688)..(699) <223> C-tag <220> <221> misc_feature <222> (700)..(702) <223> Stop codon <400> 45 atgctgagac tgctgctgag acaccacttc cactgcctgc tgctgtgtgc cgtttgggct 60 acaccttgtc tggcctctcc atggtctacc ctgaccgcca accagaatcc ttctccacct 120 tggtccaagc tgacctactc caagcctcac gatgccgcta ccttctactg cccctttctg 180 tacccatctc cacctcggag ccctctgcag ttctctggct tccagagagt gtccaccgga 240 cctgagtgcc ggaacgagac actgtacctg ctgtacaacc gcgagggcca gacactggtg 300 gaaagatcct ctacctgggt caagaaagtg atctggtatc tgagcggccg gaaccagacc 360 atcctgcaga gaatgcctcg gaccgcctct aagccttctg acggcaacgt gcagatctcc 420 gtggaagatg ccaagatctt cggcgcccac atggtgccca agcagaccaa actgctgaga 480 ttcgtggtca acgacggcac ccgctaccag atgtgcgtga tgaagctgga aagctgggcc 540 cacgtgttcc gggattactc cgtgtctttc caagtgcggc tgaccttcac cgaggccaac 600 aaccagacct acaccttctg cacccatcct aacctgatcg tcggaggcgg aggatctggc 660 ggaggtggaa gtggcggagg cggatctgag cccgaggcct aa 702 <210> 46 <211> 390 <212> DNA <213> Artificial Sequence <220> <223> UL131A (without introns) optimized for CHO expression <220> <221> misc_feature <222> (1)..(54) <223> UL131 signal peptide <220> <221> misc_feature <222> (55)..(387) <223> UL131 ectodomain <220> <221> misc_feature <222> (388)..(390) <223> Stop codon <400> 46 atgagactgt gcagagtgtg gctgtccgtg tgcctgtgtg ctgtggttct gggccagtgc 60 cagagagaga cagccgagaa gaacgactac tacagagtgc cccactactg ggacgcctgc 120 agtagagctt tgcccgatca gacccggtac aaatacgtgg aacagctggt ggatctgacc 180 ctgaactacc actacgacgc ctctcacggc ctggacaact tcgacgtgct gaagcggatc 240 aacgtgaccg aggtgtccct gctgatctct gacttccggc ggcagaatag aagaggcggc 300 accaacaagc ggaccacctt taatgctgcc ggctctctgg ctccccacgc cagatctctg 360 gaattttccg tgcggctgtt cgccaactaa 390 <210> 47 <211> 1587 <212> DNA <213> Artificial Sequence <220> <223> RSV-F-SpyTag-Ctag <220> <221> misc_feature <222> (1)..(75) <223> Signal peptide <220> <221> misc_feature <222> (1435)..(1515) <223> Foldon domain <220> <221> misc_feature <222> (1516)..(1533) <223> (GSG)2 linker <220> <221> misc_feature <222> (1534)..(1572) <223> Spytag <220> <221> misc_feature <222> (1573)..(1584) <223> Ctag <220> <221> misc_feature <222> (1585)..(1587) <223> Stop codon <400> 47 atggaactgc tgatcctgaa ggccaacgcc atcaccacca tcctgaccgc cgtgaccttc 60 tgcttcgcca gcggccagaa catcaccgag gaattctacc agagcacctg cagcgccgtg 120 agcaagggct acctgagcgc cctgcggacc ggctggtaca ccagcgtgat caccatcgag 180 ctgtccaaca tcaaagaaaa caagtgcaac ggcaccgacg ccaaagtgaa gctgatcaag 240 caggaactgg acaagtacaa gaacgccgtg accgagctgc agctgctgat gcagagcacc 300 cccgccaccg gatctggcag cgccatttgc agcggcgtgg ccgtgtgtaa agtgctgcac 360 ctggaaggcg aagtgaacaa gatcaagtcc gccctgctgt ccaccaacaa ggccgtggtg 420 tccctgagca acggcgtgag cgtgctgacc ttcaaggtgc tggatctgaa gaactacatc 480 gacaagcagc tgctgcccat cctgaacaag cagagctgca gcatcagcaa catcgagaca 540 gtgatcgagt tccagcagaa gaacaaccgg ctgctggaaa tcacccggga gttcagcgtg 600 aacgccggag tgaccacccc cgtgtccacc tacatgctga ccaacagcga gctgctgtcc 660 ctgatcaatg acatgcccat caccaacgac cagaaaaagc tgatgagcaa caacgtgcag 720 atcgtgcggc agcagagcta ctccatcatg tgcatcatca aagaagaggt gctggcctac 780 gtggtgcagc tgcccctgta cggcgtgatc gacaccccct gctggaagct gcacaccagc 840 cccctgtgca caaccaacac caaagagggc agcaacatct gcctgacccg gaccgaccgg 900 ggctggtact gcgacaacgc cggcagcgtg tccttctttc cacaggccga gacatgcaag 960 gtgcagagca accgggtgtt ctgcgacacc atgaacagcc ggaccctgcc ctccgaagtg 1020 aacctgtgca acgtggacat cttcaacccc aagtacgact gcaagatcat gacctccaag 1080 accgacgtgt ccagctccgt gatcacctcc ctgggcgcca tcgtgtcctg ctacggcaag 1140 accaagtgca ccgccagcaa caagaacaga ggcatcatca agaccttcag caacggctgc 1200 gactacgtgt ccaataaggg cgtggacacc gtgtccgtgg gcaacacact gtactgcgtg 1260 aataagcagg aaggcaagag cctgtacgtg aagggcgagc ccatcatcaa cttctacgac 1320 cccctggtgt tccccagcga cgagttcgac gctagcatca gccaggtgaa cgagaagatc 1380 aaccagagcc tggccttcat cagaaagagc gacgaactgc tgtccgccat cggcggctac 1440 atccccgagg cccccagaga tggccaggcc tacgtgcgga aggacggcga gtgggtgctg 1500 ctgtctacat ttctgggaag cggaggctct ggtgcccata tcgtgatggt ggacgcctac 1560 aagcctacca aagagcccga ggcctaa 1587 <210> 48 <211> 1515 <212> DNA <213> Artificial Sequence <220> <223> Sc9-10 DS-Cav1 A149C Y458C (RSV-F) <220> <221> misc_feature <222> (1)..(75) <223> Signal peptide <220> <221> misc_feature <222> (1435)..(1515) <223> Foldon domain <400> 48 atggaactgc tgatcctgaa ggccaacgcc atcaccacca tcctgaccgc cgtgaccttc 60 tgcttcgcca gcggccagaa catcaccgag gaattctacc agagcacctg cagcgccgtg 120 agcaagggct acctgagcgc cctgcggacc ggctggtaca ccagcgtgat caccatcgag 180 ctgtccaaca tcaaagaaaa caagtgcaac ggcaccgacg ccaaagtgaa gctgatcaag 240 caggaactgg acaagtacaa gaacgccgtg accgagctgc agctgctgat gcagagcacc 300 cccgccaccg gatctggcag cgccatttgc agcggcgtgg ccgtgtgtaa agtgctgcac 360 ctggaaggcg aagtgaacaa gatcaagtcc gccctgctgt ccaccaacaa ggccgtggtg 420 tccctgagca acggcgtgag cgtgctgacc ttcaaggtgc tggatctgaa gaactacatc 480 gacaagcagc tgctgcccat cctgaacaag cagagctgca gcatcagcaa catcgagaca 540 gtgatcgagt tccagcagaa gaacaaccgg ctgctggaaa tcacccggga gttcagcgtg 600 aacgccggag tgaccacccc cgtgtccacc tacatgctga ccaacagcga gctgctgtcc 660 ctgatcaatg acatgcccat caccaacgac cagaaaaagc tgatgagcaa caacgtgcag 720 atcgtgcggc agcagagcta ctccatcatg tgcatcatca aagaagaggt gctggcctac 780 gtggtgcagc tgcccctgta cggcgtgatc gacaccccct gctggaagct gcacaccagc 840 cccctgtgca caaccaacac caaagagggc agcaacatct gcctgacccg gaccgaccgg 900 ggctggtact gcgacaacgc cggcagcgtg tccttctttc cacaggccga gacatgcaag 960 gtgcagagca accgggtgtt ctgcgacacc atgaacagcc ggaccctgcc ctccgaagtg 1020 aacctgtgca acgtggacat cttcaacccc aagtacgact gcaagatcat gacctccaag 1080 accgacgtgt ccagctccgt gatcacctcc ctgggcgcca tcgtgtcctg ctacggcaag 1140 accaagtgca ccgccagcaa caagaacaga ggcatcatca agaccttcag caacggctgc 1200 gactacgtgt ccaataaggg cgtggacacc gtgtccgtgg gcaacacact gtactgcgtg 1260 aataagcagg aaggcaagag cctgtacgtg aagggcgagc ccatcatcaa cttctacgac 1320 cccctggtgt tccccagcga cgagttcgac gctagcatca gccaggtgaa cgagaagatc 1380 aaccagagcc tggccttcat cagaaagagc gacgaactgc tgtccgccat cggcggctac 1440 atccccgagg cccccagaga tggccaggcc tacgtgcgga aggacggcga gtgggtgctg 1500 ctgtctacat ttctg 1515 <210> 49 <211> 1440 <212> DNA <213> Artificial Sequence <220> <223> Sc-9-10 DS-Cav1 A149C Y458C (RSV-F) without the signal peptide <220> <221> misc_feature <222> (1360)..(1440) <223> Foldon domain <400> 49 cagaacatca ccgaggaatt ctaccagagc acctgcagcg ccgtgagcaa gggctacctg 60 agcgccctgc ggaccggctg gtacaccagc gtgatcacca tcgagctgtc caacatcaaa 120 gaaaacaagt gcaacggcac cgacgccaaa gtgaagctga tcaagcagga actggacaag 180 tacaagaacg ccgtgaccga gctgcagctg ctgatgcaga gcacccccgc caccggatct 240 ggcagcgcca tttgcagcgg cgtggccgtg tgtaaagtgc tgcacctgga aggcgaagtg 300 aacaagatca agtccgccct gctgtccacc aacaaggccg tggtgtccct gagcaacggc 360 gtgagcgtgc tgaccttcaa ggtgctggat ctgaagaact acatcgacaa gcagctgctg 420 cccatcctga acaagcagag ctgcagcatc agcaacatcg agacagtgat cgagttccag 480 cagaagaaca accggctgct ggaaatcacc cgggagttca gcgtgaacgc cggagtgacc 540 acccccgtgt ccacctacat gctgaccaac agcgagctgc tgtccctgat caatgacatg 600 cccatcacca acgaccagaa aaagctgatg agcaacaacg tgcagatcgt gcggcagcag 660 agctactcca tcatgtgcat catcaaagaa gaggtgctgg cctacgtggt gcagctgccc 720 ctgtacggcg tgatcgacac cccctgctgg aagctgcaca ccagccccct gtgcacaacc 780 aacaccaaag agggcagcaa catctgcctg acccggaccg accggggctg gtactgcgac 840 aacgccggca gcgtgtcctt ctttccacag gccgagacat gcaaggtgca gagcaaccgg 900 gtgttctgcg acaccatgaa cagccggacc ctgccctccg aagtgaacct gtgcaacgtg 960 gacatcttca accccaagta cgactgcaag atcatgacct ccaagaccga cgtgtccagc 1020 tccgtgatca cctccctggg cgccatcgtg tcctgctacg gcaagaccaa gtgcaccgcc 1080 agcaacaaga acagaggcat catcaagacc ttcagcaacg gctgcgacta cgtgtccaat 1140 aagggcgtgg acaccgtgtc cgtgggcaac acactgtact gcgtgaataa gcaggaaggc 1200 aagagcctgt acgtgaaggg cgagcccatc atcaacttct acgaccccct ggtgttcccc 1260 agcgacgagt tcgacgctag catcagccag gtgaacgaga agatcaacca gagcctggcc 1320 ttcatcagaa agagcgacga actgctgtcc gccatcggcg gctacatccc cgaggccccc 1380 agagatggcc aggcctacgt gcggaaggac ggcgagtggg tgctgctgtc tacatttctg 1440 <210> 50 <211> 528 <212> PRT <213> Artificial Sequence <220> <223> RSV-F-SpyTag-Ctag <220> <221> MISC_FEATURE <222> (1)..(25) <223> Signal peptide <220> <221> MISC_FEATURE <222> (479)..(505) <223> Foldon domain <220> <221> MISC_FEATURE <222> (506)..(511) <223> (GSG)2 linker <220> <221> MISC_FEATURE <222> (512)..(524) <223> Spytag <220> <221> MISC_FEATURE <222> (525)..(527) <223> Ctag <400> 50 Met Glu Leu Leu Ile Leu Lys Ala Asn Ala Ile Thr Thr Ile Leu Thr 1 5 10 15 Ala Val Thr Phe Cys Phe Ala Ser Gly Gln Asn Ile Thr Glu Glu Phe 20 25 30 Tyr Gln Ser Thr Cys Ser Ala Val Ser Lys Gly Tyr Leu Ser Ala Leu 35 40 45 Arg Thr Gly Trp Tyr Thr Ser Val Ile Thr Ile Glu Leu Ser Asn Ile 50 55 60 Lys Glu Asn Lys Cys Asn Gly Thr Asp Ala Lys Val Lys Leu Ile Lys 65 70 75 80 Gln Glu Leu Asp Lys Tyr Lys Asn Ala Val Thr Glu Leu Gln Leu Leu 85 90 95 Met Gln Ser Thr Pro Ala Thr Gly Ser Gly Ser Ala Ile Cys Ser Gly 100 105 110 Val Ala Val Cys Lys Val Leu His Leu Glu Gly Glu Val Asn Lys Ile 115 120 125 Lys Ser Ala Leu Leu Ser Thr Asn Lys Ala Val Val Ser Leu Ser Asn 130 135 140 Gly Val Ser Val Leu Thr Phe Lys Val Leu Asp Leu Lys Asn Tyr Ile 145 150 155 160 Asp Lys Gln Leu Leu Pro Ile Leu Asn Lys Gln Ser Cys Ser Ile Ser 165 170 175 Asn Ile Glu Thr Val Ile Glu Phe Gln Gln Lys Asn Asn Arg Leu Leu 180 185 190 Glu Ile Thr Arg Glu Phe Ser Val Asn Ala Gly Val Thr Thr Pro Val 195 200 205 Ser Thr Tyr Met Leu Thr Asn Ser Glu Leu Leu Ser Leu Ile Asn Asp 210 215 220 Met Pro Ile Thr Asn Asp Gln Lys Lys Leu Met Ser Asn Asn Val Gln 225 230 235 240 Ile Val Arg Gln Gln Ser Tyr Ser Ile Met Cys Ile Ile Lys Glu Glu 245 250 255 Val Leu Ala Tyr Val Val Gln Leu Pro Leu Tyr Gly Val Ile Asp Thr 260 265 270 Pro Cys Trp Lys Leu His Thr Ser Pro Leu Cys Thr Thr Asn Thr Lys 275 280 285 Glu Gly Ser Asn Ile Cys Leu Thr Arg Thr Asp Arg Gly Trp Tyr Cys 290 295 300 Asp Asn Ala Gly Ser Val Ser Phe Phe Pro Gln Ala Glu Thr Cys Lys 305 310 315 320 Val Gln Ser Asn Arg Val Phe Cys Asp Thr Met Asn Ser Arg Thr Leu 325 330 335 Pro Ser Glu Val Asn Leu Cys Asn Val Asp Ile Phe Asn Pro Lys Tyr 340 345 350 Asp Cys Lys Ile Met Thr Ser Lys Thr Asp Val Ser Ser Ser Val Ile 355 360 365 Thr Ser Leu Gly Ala Ile Val Ser Cys Tyr Gly Lys Thr Lys Cys Thr 370 375 380 Ala Ser Asn Lys Asn Arg Gly Ile Ile Lys Thr Phe Ser Asn Gly Cys 385 390 395 400 Asp Tyr Val Ser Asn Lys Gly Val Asp Thr Val Ser Val Gly Asn Thr 405 410 415 Leu Tyr Cys Val Asn Lys Gln Glu Gly Lys Ser Leu Tyr Val Lys Gly 420 425 430 Glu Pro Ile Ile Asn Phe Tyr Asp Pro Leu Val Phe Pro Ser Asp Glu 435 440 445 Phe Asp Ala Ser Ile Ser Gln Val Asn Glu Lys Ile Asn Gln Ser Leu 450 455 460 Ala Phe Ile Arg Lys Ser Asp Glu Leu Leu Ser Ala Ile Gly Gly Tyr 465 470 475 480 Ile Pro Glu Ala Pro Arg Asp Gly Gln Ala Tyr Val Arg Lys Asp Gly 485 490 495 Glu Trp Val Leu Leu Ser Thr Phe Leu Gly Ser Gly Gly Ser Gly Ala 500 505 510 His Ile Val Met Val Asp Ala Tyr Lys Pro Thr Lys Glu Pro Glu Ala 515 520 525 <210> 51 <211> 1010 <212> PRT <213> Artificial Sequence <220> <223> Sc-9-10 DS-Cav1 A149C Y458C (RSV-F) <220> <221> MISC_FEATURE <222> (1)..(25) <223> Signal peptide <220> <221> MISC_FEATURE <222> (479)..(505) <223> Foldon domain <400> 51 Met Glu Leu Leu Ile Leu Lys Ala Asn Ala Ile Thr Thr Ile Leu Thr 1 5 10 15 Ala Val Thr Phe Cys Phe Ala Ser Gly Gln Asn Ile Thr Glu Glu Phe 20 25 30 Tyr Gln Ser Thr Cys Ser Ala Val Ser Lys Gly Tyr Leu Ser Ala Leu 35 40 45 Arg Thr Gly Trp Tyr Thr Ser Val Ile Thr Ile Glu Leu Ser Asn Ile 50 55 60 Lys Glu Asn Lys Cys Asn Gly Thr Asp Ala Lys Val Lys Leu Ile Lys 65 70 75 80 Gln Glu Leu Asp Lys Tyr Lys Asn Ala Val Thr Glu Leu Gln Leu Leu 85 90 95 Met Gln Ser Thr Pro Ala Thr Gly Ser Gly Ser Ala Ile Cys Ser Gly 100 105 110 Val Ala Val Cys Lys Val Leu His Leu Glu Gly Glu Val Asn Lys Ile 115 120 125 Lys Ser Ala Leu Leu Ser Thr Asn Lys Ala Val Val Ser Leu Ser Asn 130 135 140 Gly Val Ser Val Leu Thr Phe Lys Val Leu Asp Leu Lys Asn Tyr Ile 145 150 155 160 Asp Lys Gln Leu Leu Pro Ile Leu Asn Lys Gln Ser Cys Ser Ile Ser 165 170 175 Asn Ile Glu Thr Val Ile Glu Phe Gln Gln Lys Asn Asn Arg Leu Leu 180 185 190 Glu Ile Thr Arg Glu Phe Ser Val Asn Ala Gly Val Thr Thr Pro Val 195 200 205 Ser Thr Tyr Met Leu Thr Asn Ser Glu Leu Leu Ser Leu Ile Asn Asp 210 215 220 Met Pro Ile Thr Asn Asp Gln Lys Lys Leu Met Ser Asn Asn Val Gln 225 230 235 240 Ile Val Arg Gln Gln Ser Tyr Ser Ile Met Cys Ile Ile Lys Glu Glu 245 250 255 Val Leu Ala Tyr Val Val Gln Leu Pro Leu Tyr Gly Val Ile Asp Thr 260 265 270 Pro Cys Trp Lys Leu His Thr Ser Pro Leu Cys Thr Thr Asn Thr Lys 275 280 285 Glu Gly Ser Asn Ile Cys Leu Thr Arg Thr Asp Arg Gly Trp Tyr Cys 290 295 300 Asp Asn Ala Gly Ser Val Ser Phe Phe Pro Gln Ala Glu Thr Cys Lys 305 310 315 320 Val Gln Ser Asn Arg Val Phe Cys Asp Thr Met Asn Ser Arg Thr Leu 325 330 335 Pro Ser Glu Val Asn Leu Cys Asn Val Asp Ile Phe Asn Pro Lys Tyr 340 345 350 Asp Cys Lys Ile Met Thr Ser Lys Thr Asp Val Ser Ser Ser Val Ile 355 360 365 Thr Ser Leu Met Glu Leu Leu Ile Leu Lys Ala Asn Ala Ile Thr Thr 370 375 380 Ile Leu Thr Ala Val Thr Phe Cys Phe Ala Ser Gly Gln Asn Ile Thr 385 390 395 400 Glu Glu Phe Tyr Gln Ser Thr Cys Ser Ala Val Ser Lys Gly Tyr Leu 405 410 415 Ser Ala Leu Arg Thr Gly Trp Tyr Thr Ser Val Ile Thr Ile Glu Leu 420 425 430 Ser Asn Ile Lys Glu Asn Lys Cys Asn Gly Thr Asp Ala Lys Val Lys 435 440 445 Leu Ile Lys Gln Glu Leu Asp Lys Tyr Lys Asn Ala Val Thr Glu Leu 450 455 460 Gln Leu Leu Met Gln Ser Thr Pro Ala Thr Gly Ser Gly Ser Ala Ile 465 470 475 480 Cys Ser Gly Val Ala Val Cys Lys Val Leu His Leu Glu Gly Glu Val 485 490 495 Asn Lys Ile Lys Ser Ala Leu Leu Ser Thr Asn Lys Ala Val Val Ser 500 505 510 Leu Ser Asn Gly Val Ser Val Leu Thr Phe Lys Val Leu Asp Leu Lys 515 520 525 Asn Tyr Ile Asp Lys Gln Leu Leu Pro Ile Leu Asn Lys Gln Ser Cys 530 535 540 Ser Ile Ser Asn Ile Glu Thr Val Ile Glu Phe Gln Gln Lys Asn Asn 545 550 555 560 Arg Leu Leu Glu Ile Thr Arg Glu Phe Ser Val Asn Ala Gly Val Thr 565 570 575 Thr Pro Val Ser Thr Tyr Met Leu Thr Asn Ser Glu Leu Leu Ser Leu 580 585 590 Ile Asn Asp Met Pro Ile Thr Asn Asp Gln Lys Lys Leu Met Ser Asn 595 600 605 Asn Val Gln Ile Val Arg Gln Gln Ser Tyr Ser Ile Met Cys Ile Ile 610 615 620 Lys Glu Glu Val Leu Ala Tyr Val Val Gln Leu Pro Leu Tyr Gly Val 625 630 635 640 Ile Asp Thr Pro Cys Trp Lys Leu His Thr Ser Pro Leu Cys Thr Thr 645 650 655 Asn Thr Lys Glu Gly Ser Asn Ile Cys Leu Thr Arg Thr Asp Arg Gly 660 665 670 Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser Phe Phe Pro Gln Ala Glu 675 680 685 Thr Cys Lys Val Gln Ser Asn Arg Val Phe Cys Asp Thr Met Asn Ser 690 695 700 Arg Thr Leu Pro Ser Glu Val Asn Leu Cys Asn Val Asp Ile Phe Asn 705 710 715 720 Pro Lys Tyr Asp Cys Lys Ile Met Thr Ser Lys Thr Asp Val Ser Ser 725 730 735 Ser Val Ile Thr Ser Leu Gly Ala Ile Val Ser Cys Tyr Gly Lys Thr 740 745 750 Lys Cys Thr Ala Ser Asn Lys Asn Arg Gly Ile Ile Lys Thr Phe Ser 755 760 765 Asn Gly Cys Asp Tyr Val Ser Asn Lys Gly Val Asp Thr Val Ser Val 770 775 780 Gly Asn Thr Leu Tyr Cys Val Asn Lys Gln Glu Gly Lys Ser Leu Tyr 785 790 795 800 Val Lys Gly Glu Pro Ile Ile Asn Phe Tyr Asp Pro Leu Val Phe Pro 805 810 815 Ser Asp Glu Phe Asp Ala Ser Ile Ser Gln Val Asn Glu Lys Ile Asn 820 825 830 Gln Ser Leu Ala Phe Ile Arg Lys Ser Asp Glu Leu Leu Ser Ala Ile 835 840 845 Gly Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln Ala Tyr Val Arg 850 855 860 Lys Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu Gly Ala Ile Val 865 870 875 880 Ser Cys Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn Lys Asn Arg Gly 885 890 895 Ile Ile Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val Ser Asn Lys Gly 900 905 910 Val Asp Thr Val Ser Val Gly Asn Thr Leu Tyr Cys Val Asn Lys Gln 915 920 925 Glu Gly Lys Ser Leu Tyr Val Lys Gly Glu Pro Ile Ile Asn Phe Tyr 930 935 940 Asp Pro Leu Val Phe Pro Ser Asp Glu Phe Asp Ala Ser Ile Ser Gln 945 950 955 960 Val Asn Glu Lys Ile Asn Gln Ser Leu Ala Phe Ile Arg Lys Ser Asp 965 970 975 Glu Leu Leu Ser Ala Ile Gly Gly Tyr Ile Pro Glu Ala Pro Arg Asp 980 985 990 Gly Gln Ala Tyr Val Arg Lys Asp Gly Glu Trp Val Leu Leu Ser Thr 995 1000 1005 Phe Leu 1010 <210> 52 <211> 480 <212> PRT <213> Artificial Sequence <220> <223> sc9-10 DS-Cav1 A149C Y458C (RSV-F) without the signal peptide <220> <221> MISC_FEATURE <222> (454)..(480) <223> Foldon domain <400> 52 Gln Asn Ile Thr Glu Glu Phe Tyr Gln Ser Thr Cys Ser Ala Val Ser 1 5 10 15 Lys Gly Tyr Leu Ser Ala Leu Arg Thr Gly Trp Tyr Thr Ser Val Ile 20 25 30 Thr Ile Glu Leu Ser Asn Ile Lys Glu Asn Lys Cys Asn Gly Thr Asp 35 40 45 Ala Lys Val Lys Leu Ile Lys Gln Glu Leu Asp Lys Tyr Lys Asn Ala 50 55 60 Val Thr Glu Leu Gln Leu Leu Met Gln Ser Thr Pro Ala Thr Gly Ser 65 70 75 80 Gly Ser Ala Ile Cys Ser Gly Val Ala Val Cys Lys Val Leu His Leu 85 90 95 Glu Gly Glu Val Asn Lys Ile Lys Ser Ala Leu Leu Ser Thr Asn Lys 100 105 110 Ala Val Val Ser Leu Ser Asn Gly Val Ser Val Leu Thr Phe Lys Val 115 120 125 Leu Asp Leu Lys Asn Tyr Ile Asp Lys Gln Leu Leu Pro Ile Leu Asn 130 135 140 Lys Gln Ser Cys Ser Ile Ser Asn Ile Glu Thr Val Ile Glu Phe Gln 145 150 155 160 Gln Lys Asn Asn Arg Leu Leu Glu Ile Thr Arg Glu Phe Ser Val Asn 165 170 175 Ala Gly Val Thr Thr Pro Val Ser Thr Tyr Met Leu Thr Asn Ser Glu 180 185 190 Leu Leu Ser Leu Ile Asn Asp Met Pro Ile Thr Asn Asp Gln Lys Lys 195 200 205 Leu Met Ser Asn Asn Val Gln Ile Val Arg Gln Gln Ser Tyr Ser Ile 210 215 220 Met Cys Ile Ile Lys Glu Glu Val Leu Ala Tyr Val Val Gln Leu Pro 225 230 235 240 Leu Tyr Gly Val Ile Asp Thr Pro Cys Trp Lys Leu His Thr Ser Pro 245 250 255 Leu Cys Thr Thr Asn Thr Lys Glu Gly Ser Asn Ile Cys Leu Thr Arg 260 265 270 Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser Phe Phe 275 280 285 Pro Gln Ala Glu Thr Cys Lys Val Gln Ser Asn Arg Val Phe Cys Asp 290 295 300 Thr Met Asn Ser Arg Thr Leu Pro Ser Glu Val Asn Leu Cys Asn Val 305 310 315 320 Asp Ile Phe Asn Pro Lys Tyr Asp Cys Lys Ile Met Thr Ser Lys Thr 325 330 335 Asp Val Ser Ser Ser Val Ile Thr Ser Leu Gly Ala Ile Val Ser Cys 340 345 350 Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn Lys Asn Arg Gly Ile Ile 355 360 365 Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val Ser Asn Lys Gly Val Asp 370 375 380 Thr Val Ser Val Gly Asn Thr Leu Tyr Cys Val Asn Lys Gln Glu Gly 385 390 395 400 Lys Ser Leu Tyr Val Lys Gly Glu Pro Ile Ile Asn Phe Tyr Asp Pro 405 410 415 Leu Val Phe Pro Ser Asp Glu Phe Asp Ala Ser Ile Ser Gln Val Asn 420 425 430 Glu Lys Ile Asn Gln Ser Leu Ala Phe Ile Arg Lys Ser Asp Glu Leu 435 440 445 Leu Ser Ala Ile Gly Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln 450 455 460 Ala Tyr Val Arg Lys Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu 465 470 475 480 <210> 53 <211> 1704 <212> DNA <213> Artificial Sequence <220> <223> RSV-F DS-Cav1-SpyTag-Ctag <220> <221> misc_feature <222> (1)..(75) <223> Signal peptide <220> <221> misc_feature <222> (1552)..(1632) <223> Foldon domain <220> <221> misc_feature <222> (1633)..(1650) <223> (GSG)2 linker <220> <221> misc_feature <222> (1651)..(1689) <223> Spytag <220> <221> misc_feature <222> (1690)..(1701) <223> Ctag <220> <221> misc_feature <222> (1702)..(1704) <223> Stop codon <400> 53 atggaactgc tgatcctgaa ggccaacgcc atcaccacca tcctgaccgc cgtgaccttc 60 tgcttcgcca gcggccagaa catcaccgag gaattctacc agagcacctg cagcgccgtg 120 agcaagggct acctgagcgc cctgcggacc ggctggtaca ccagcgtgat caccatcgag 180 ctgtccaaca tcaaagaaaa caagtgcaac ggcaccgacg ccaaagtgaa gctgatcaag 240 caggaactgg acaagtacaa gaacgccgtg accgagctgc agctgctgat gcagagcacc 300 cccgccacca acaacagagc cagaagagag ctgccccggt tcatgaacta caccctgaac 360 aacgccaaga aaaccaacgt gaccctgagc aagaagagaa agagaagatt cctgggcttc 420 ctgctgggcg tgggcagcgc cattgccagc ggcgtggccg tgtgtaaagt gctgcacctg 480 gaaggcgaag tgaacaagat caagtccgcc ctgctgtcca ccaacaaggc cgtggtgtcc 540 ctgagcaacg gcgtgagcgt gctgaccttc aaggtgctgg atctgaagaa ctacatcgac 600 aagcagctgc tgcccatcct gaacaagcag agctgcagca tcagcaacat cgagacagtg 660 atcgagttcc agcagaagaa caaccggctg ctggaaatca cccgggagtt cagcgtgaac 720 gccggagtga ccacccccgt gtccacctac atgctgacca acagcgagct gctgtccctg 780 atcaatgaca tgcccatcac caacgaccag aaaaagctga tgagcaacaa cgtgcagatc 840 gtgcggcagc agagctactc catcatgtgc atcatcaaag aagaggtgct ggcctacgtg 900 gtgcagctgc ccctgtacgg cgtgatcgac accccctgct ggaagctgca caccagcccc 960 ctgtgcacaa ccaacaccaa agagggcagc aacatctgcc tgacccggac cgaccggggc 1020 tggtactgcg acaacgccgg cagcgtgtcc ttctttccac aggccgagac atgcaaggtg 1080 cagagcaacc gggtgttctg cgacaccatg aacagcctga ccctgccctc cgaagtgaac 1140 ctgtgcaacg tggacatctt caaccccaag tacgactgca agatcatgac ctccaagacc 1200 gacgtgtcca gctccgtgat cacctccctg ggcgccatcg tgtcctgcta cggcaagacc 1260 aagtgcaccg ccagcaacaa gaacagaggc atcatcaaga ccttcagcaa cggctgcgac 1320 tacgtgtcca ataagggcgt ggacaccgtg tccgtgggca acacactgta ctacgtgaat 1380 aagcaggaag gcaagagcct gtacgtgaag ggcgagccca tcatcaactt ctacgacccc 1440 ctggtgttcc ccagcgacga gttcgacgcc agcatcagcc aggtgaacga gaagatcaac 1500 cagagcctgg ccttcatcag aaagagcgac gaactgctgt ccgccatcgg cggctacatc 1560 cccgaggccc ccagagatgg ccaggcctac gtgcggaagg acggcgagtg ggtgctgctg 1620 tctacatttc tgggaagcgg aggctctggt gcccatatcg tgatggtgga cgcctacaag 1680 cctaccaaag agcccgaggc ctaa 1704 <210> 54 <211> 1632 <212> DNA <213> Artificial Sequence <220> <223> RSV-F DS-Cav1 <220> <221> misc_feature <222> (1)..(75) <223> Signal peptide <220> <221> misc_feature <222> (1552)..(1632) <223> Foldon domain <400> 54 atggaactgc tgatcctgaa ggccaacgcc atcaccacca tcctgaccgc cgtgaccttc 60 tgcttcgcca gcggccagaa catcaccgag gaattctacc agagcacctg cagcgccgtg 120 agcaagggct acctgagcgc cctgcggacc ggctggtaca ccagcgtgat caccatcgag 180 ctgtccaaca tcaaagaaaa caagtgcaac ggcaccgacg ccaaagtgaa gctgatcaag 240 caggaactgg acaagtacaa gaacgccgtg accgagctgc agctgctgat gcagagcacc 300 cccgccacca acaacagagc cagaagagag ctgccccggt tcatgaacta caccctgaac 360 aacgccaaga aaaccaacgt gaccctgagc aagaagagaa agagaagatt cctgggcttc 420 ctgctgggcg tgggcagcgc cattgccagc ggcgtggccg tgtgtaaagt gctgcacctg 480 gaaggcgaag tgaacaagat caagtccgcc ctgctgtcca ccaacaaggc cgtggtgtcc 540 ctgagcaacg gcgtgagcgt gctgaccttc aaggtgctgg atctgaagaa ctacatcgac 600 aagcagctgc tgcccatcct gaacaagcag agctgcagca tcagcaacat cgagacagtg 660 atcgagttcc agcagaagaa caaccggctg ctggaaatca cccgggagtt cagcgtgaac 720 gccggagtga ccacccccgt gtccacctac atgctgacca acagcgagct gctgtccctg 780 atcaatgaca tgcccatcac caacgaccag aaaaagctga tgagcaacaa cgtgcagatc 840 gtgcggcagc agagctactc catcatgtgc atcatcaaag aagaggtgct ggcctacgtg 900 gtgcagctgc ccctgtacgg cgtgatcgac accccctgct ggaagctgca caccagcccc 960 ctgtgcacaa ccaacaccaa agagggcagc aacatctgcc tgacccggac cgaccggggc 1020 tggtactgcg acaacgccgg cagcgtgtcc ttctttccac aggccgagac atgcaaggtg 1080 cagagcaacc gggtgttctg cgacaccatg aacagcctga ccctgccctc cgaagtgaac 1140 ctgtgcaacg tggacatctt caaccccaag tacgactgca agatcatgac ctccaagacc 1200 gacgtgtcca gctccgtgat cacctccctg ggcgccatcg tgtcctgcta cggcaagacc 1260 aagtgcaccg ccagcaacaa gaacagaggc atcatcaaga ccttcagcaa cggctgcgac 1320 tacgtgtcca ataagggcgt ggacaccgtg tccgtgggca acacactgta ctacgtgaat 1380 aagcaggaag gcaagagcct gtacgtgaag ggcgagccca tcatcaactt ctacgacccc 1440 ctggtgttcc ccagcgacga gttcgacgcc agcatcagcc aggtgaacga gaagatcaac 1500 cagagcctgg ccttcatcag aaagagcgac gaactgctgt ccgccatcgg cggctacatc 1560 cccgaggccc ccagagatgg ccaggcctac gtgcggaagg acggcgagtg ggtgctgctg 1620 tctacatttc tg 1632 <210> 55 <211> 1557 <212> DNA <213> Artificial Sequence <220> <223> RSV-F DS-Cav1 without the signal peptide <220> <221> misc_feature <222> (1477)..(1557) <223> Foldon domain <400> 55 cagaacatca ccgaggaatt ctaccagagc acctgcagcg ccgtgagcaa gggctacctg 60 agcgccctgc ggaccggctg gtacaccagc gtgatcacca tcgagctgtc caacatcaaa 120 gaaaacaagt gcaacggcac cgacgccaaa gtgaagctga tcaagcagga actggacaag 180 tacaagaacg ccgtgaccga gctgcagctg ctgatgcaga gcacccccgc caccaacaac 240 agagccagaa gagagctgcc ccggttcatg aactacaccc tgaacaacgc caagaaaacc 300 aacgtgaccc tgagcaagaa gagaaagaga agattcctgg gcttcctgct gggcgtgggc 360 agcgccattg ccagcggcgt ggccgtgtgt aaagtgctgc acctggaagg cgaagtgaac 420 aagatcaagt ccgccctgct gtccaccaac aaggccgtgg tgtccctgag caacggcgtg 480 agcgtgctga ccttcaaggt gctggatctg aagaactaca tcgacaagca gctgctgccc 540 atcctgaaca agcagagctg cagcatcagc aacatcgaga cagtgatcga gttccagcag 600 aagaacaacc ggctgctgga aatcacccgg gagttcagcg tgaacgccgg agtgaccacc 660 cccgtgtcca cctacatgct gaccaacagc gagctgctgt ccctgatcaa tgacatgccc 720 atcaccaacg accagaaaaa gctgatgagc aacaacgtgc agatcgtgcg gcagcagagc 780 tactccatca tgtgcatcat caaagaagag gtgctggcct acgtggtgca gctgcccctg 840 tacggcgtga tcgacacccc ctgctggaag ctgcacacca gccccctgtg cacaaccaac 900 accaaagagg gcagcaacat ctgcctgacc cggaccgacc ggggctggta ctgcgacaac 960 gccggcagcg tgtccttctt tccacaggcc gagacatgca aggtgcagag caaccgggtg 1020 ttctgcgaca ccatgaacag cctgaccctg ccctccgaag tgaacctgtg caacgtggac 1080 atcttcaacc ccaagtacga ctgcaagatc atgacctcca agaccgacgt gtccagctcc 1140 gtgatcacct ccctgggcgc catcgtgtcc tgctacggca agaccaagtg caccgccagc 1200 aacaagaaca gaggcatcat caagaccttc agcaacggct gcgactacgt gtccaataag 1260 ggcgtggaca ccgtgtccgt gggcaacaca ctgtactacg tgaataagca ggaaggcaag 1320 agcctgtacg tgaagggcga gcccatcatc aacttctacg accccctggt gttccccagc 1380 gacgagttcg acgccagcat cagccaggtg aacgagaaga tcaaccagag cctggccttc 1440 atcagaaaga gcgacgaact gctgtccgcc atcggcggct acatccccga ggcccccaga 1500 gatggccagg cctacgtgcg gaaggacggc gagtgggtgc tgctgtctac atttctg 1557 <210> 56 <211> 567 <212> PRT <213> Artificial Sequence <220> <223> RSV-F DS-Cav1-SpyTag-Ctag <220> <221> MISC_FEATURE <222> (1)..(25) <223> Signal peptide <220> <221> MISC_FEATURE <222> (518)..(544) <223> Foldon domain <220> <221> MISC_FEATURE <222> (545)..(550) <223> Linker <220> <221> MISC_FEATURE <222> (551)..(563) <223> Spytag <220> <221> MISC_FEATURE <222> (564)..(567) <223> Ctag <400> 56 Met Glu Leu Leu Ile Leu Lys Ala Asn Ala Ile Thr Thr Ile Leu Thr 1 5 10 15 Ala Val Thr Phe Cys Phe Ala Ser Gly Gln Asn Ile Thr Glu Glu Phe 20 25 30 Tyr Gln Ser Thr Cys Ser Ala Val Ser Lys Gly Tyr Leu Ser Ala Leu 35 40 45 Arg Thr Gly Trp Tyr Thr Ser Val Ile Thr Ile Glu Leu Ser Asn Ile 50 55 60 Lys Glu Asn Lys Cys Asn Gly Thr Asp Ala Lys Val Lys Leu Ile Lys 65 70 75 80 Gln Glu Leu Asp Lys Tyr Lys Asn Ala Val Thr Glu Leu Gln Leu Leu 85 90 95 Met Gln Ser Thr Pro Ala Thr Asn Asn Arg Ala Arg Arg Glu Leu Pro 100 105 110 Arg Phe Met Asn Tyr Thr Leu Asn Asn Ala Lys Lys Thr Asn Val Thr 115 120 125 Leu Ser Lys Lys Arg Lys Arg Arg Phe Leu Gly Phe Leu Leu Gly Val 130 135 140 Gly Ser Ala Ile Ala Ser Gly Val Ala Val Cys Lys Val Leu His Leu 145 150 155 160 Glu Gly Glu Val Asn Lys Ile Lys Ser Ala Leu Leu Ser Thr Asn Lys 165 170 175 Ala Val Val Ser Leu Ser Asn Gly Val Ser Val Leu Thr Phe Lys Val 180 185 190 Leu Asp Leu Lys Asn Tyr Ile Asp Lys Gln Leu Leu Pro Ile Leu Asn 195 200 205 Lys Gln Ser Cys Ser Ile Ser Asn Ile Glu Thr Val Ile Glu Phe Gln 210 215 220 Gln Lys Asn Asn Arg Leu Leu Glu Ile Thr Arg Glu Phe Ser Val Asn 225 230 235 240 Ala Gly Val Thr Thr Pro Val Ser Thr Tyr Met Leu Thr Asn Ser Glu 245 250 255 Leu Leu Ser Leu Ile Asn Asp Met Pro Ile Thr Asn Asp Gln Lys Lys 260 265 270 Leu Met Ser Asn Asn Val Gln Ile Val Arg Gln Gln Ser Tyr Ser Ile 275 280 285 Met Cys Ile Ile Lys Glu Glu Val Leu Ala Tyr Val Val Gln Leu Pro 290 295 300 Leu Tyr Gly Val Ile Asp Thr Pro Cys Trp Lys Leu His Thr Ser Pro 305 310 315 320 Leu Cys Thr Thr Asn Thr Lys Glu Gly Ser Asn Ile Cys Leu Thr Arg 325 330 335 Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser Phe Phe 340 345 350 Pro Gln Ala Glu Thr Cys Lys Val Gln Ser Asn Arg Val Phe Cys Asp 355 360 365 Thr Met Asn Ser Leu Thr Leu Pro Ser Glu Val Asn Leu Cys Asn Val 370 375 380 Asp Ile Phe Asn Pro Lys Tyr Asp Cys Lys Ile Met Thr Ser Lys Thr 385 390 395 400 Asp Val Ser Ser Ser Val Ile Thr Ser Leu Gly Ala Ile Val Ser Cys 405 410 415 Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn Lys Asn Arg Gly Ile Ile 420 425 430 Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val Ser Asn Lys Gly Val Asp 435 440 445 Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr Val Asn Lys Gln Glu Gly 450 455 460 Lys Ser Leu Tyr Val Lys Gly Glu Pro Ile Ile Asn Phe Tyr Asp Pro 465 470 475 480 Leu Val Phe Pro Ser Asp Glu Phe Asp Ala Ser Ile Ser Gln Val Asn 485 490 495 Glu Lys Ile Asn Gln Ser Leu Ala Phe Ile Arg Lys Ser Asp Glu Leu 500 505 510 Leu Ser Ala Ile Gly Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln 515 520 525 Ala Tyr Val Arg Lys Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu 530 535 540 Gly Ser Gly Gly Ser Gly Ala His Ile Val Met Val Asp Ala Tyr Lys 545 550 555 560 Pro Thr Lys Glu Pro Glu Ala 565 <210> 57 <211> 544 <212> PRT <213> Artificial Sequence <220> <223> RSV-F DS-Cav1 <220> <221> MISC_FEATURE <222> (1)..(25) <223> Signal peptide <220> <221> MISC_FEATURE <222> (518)..(544) <223> Foldon domain <400> 57 Met Glu Leu Leu Ile Leu Lys Ala Asn Ala Ile Thr Thr Ile Leu Thr 1 5 10 15 Ala Val Thr Phe Cys Phe Ala Ser Gly Gln Asn Ile Thr Glu Glu Phe 20 25 30 Tyr Gln Ser Thr Cys Ser Ala Val Ser Lys Gly Tyr Leu Ser Ala Leu 35 40 45 Arg Thr Gly Trp Tyr Thr Ser Val Ile Thr Ile Glu Leu Ser Asn Ile 50 55 60 Lys Glu Asn Lys Cys Asn Gly Thr Asp Ala Lys Val Lys Leu Ile Lys 65 70 75 80 Gln Glu Leu Asp Lys Tyr Lys Asn Ala Val Thr Glu Leu Gln Leu Leu 85 90 95 Met Gln Ser Thr Pro Ala Thr Asn Asn Arg Ala Arg Arg Glu Leu Pro 100 105 110 Arg Phe Met Asn Tyr Thr Leu Asn Asn Ala Lys Lys Thr Asn Val Thr 115 120 125 Leu Ser Lys Lys Arg Lys Arg Arg Phe Leu Gly Phe Leu Leu Gly Val 130 135 140 Gly Ser Ala Ile Ala Ser Gly Val Ala Val Cys Lys Val Leu His Leu 145 150 155 160 Glu Gly Glu Val Asn Lys Ile Lys Ser Ala Leu Leu Ser Thr Asn Lys 165 170 175 Ala Val Val Ser Leu Ser Asn Gly Val Ser Val Leu Thr Phe Lys Val 180 185 190 Leu Asp Leu Lys Asn Tyr Ile Asp Lys Gln Leu Leu Pro Ile Leu Asn 195 200 205 Lys Gln Ser Cys Ser Ile Ser Asn Ile Glu Thr Val Ile Glu Phe Gln 210 215 220 Gln Lys Asn Asn Arg Leu Leu Glu Ile Thr Arg Glu Phe Ser Val Asn 225 230 235 240 Ala Gly Val Thr Thr Pro Val Ser Thr Tyr Met Leu Thr Asn Ser Glu 245 250 255 Leu Leu Ser Leu Ile Asn Asp Met Pro Ile Thr Asn Asp Gln Lys Lys 260 265 270 Leu Met Ser Asn Asn Val Gln Ile Val Arg Gln Gln Ser Tyr Ser Ile 275 280 285 Met Cys Ile Ile Lys Glu Glu Val Leu Ala Tyr Val Val Gln Leu Pro 290 295 300 Leu Tyr Gly Val Ile Asp Thr Pro Cys Trp Lys Leu His Thr Ser Pro 305 310 315 320 Leu Cys Thr Thr Asn Thr Lys Glu Gly Ser Asn Ile Cys Leu Thr Arg 325 330 335 Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser Phe Phe 340 345 350 Pro Gln Ala Glu Thr Cys Lys Val Gln Ser Asn Arg Val Phe Cys Asp 355 360 365 Thr Met Asn Ser Leu Thr Leu Pro Ser Glu Val Asn Leu Cys Asn Val 370 375 380 Asp Ile Phe Asn Pro Lys Tyr Asp Cys Lys Ile Met Thr Ser Lys Thr 385 390 395 400 Asp Val Ser Ser Ser Val Ile Thr Ser Leu Gly Ala Ile Val Ser Cys 405 410 415 Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn Lys Asn Arg Gly Ile Ile 420 425 430 Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val Ser Asn Lys Gly Val Asp 435 440 445 Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr Val Asn Lys Gln Glu Gly 450 455 460 Lys Ser Leu Tyr Val Lys Gly Glu Pro Ile Ile Asn Phe Tyr Asp Pro 465 470 475 480 Leu Val Phe Pro Ser Asp Glu Phe Asp Ala Ser Ile Ser Gln Val Asn 485 490 495 Glu Lys Ile Asn Gln Ser Leu Ala Phe Ile Arg Lys Ser Asp Glu Leu 500 505 510 Leu Ser Ala Ile Gly Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln 515 520 525 Ala Tyr Val Arg Lys Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu 530 535 540 <210> 58 <211> 519 <212> PRT <213> Artificial Sequence <220> <223> RSV-F DS-Cav1 without the signal peptide <220> <221> MISC_FEATURE <222> (493)..(519) <223> Foldon domain <400> 58 Gln Asn Ile Thr Glu Glu Phe Tyr Gln Ser Thr Cys Ser Ala Val Ser 1 5 10 15 Lys Gly Tyr Leu Ser Ala Leu Arg Thr Gly Trp Tyr Thr Ser Val Ile 20 25 30 Thr Ile Glu Leu Ser Asn Ile Lys Glu Asn Lys Cys Asn Gly Thr Asp 35 40 45 Ala Lys Val Lys Leu Ile Lys Gln Glu Leu Asp Lys Tyr Lys Asn Ala 50 55 60 Val Thr Glu Leu Gln Leu Leu Met Gln Ser Thr Pro Ala Thr Asn Asn 65 70 75 80 Arg Ala Arg Arg Glu Leu Pro Arg Phe Met Asn Tyr Thr Leu Asn Asn 85 90 95 Ala Lys Lys Thr Asn Val Thr Leu Ser Lys Lys Arg Lys Arg Arg Phe 100 105 110 Leu Gly Phe Leu Leu Gly Val Gly Ser Ala Ile Ala Ser Gly Val Ala 115 120 125 Val Cys Lys Val Leu His Leu Glu Gly Glu Val Asn Lys Ile Lys Ser 130 135 140 Ala Leu Leu Ser Thr Asn Lys Ala Val Val Ser Leu Ser Asn Gly Val 145 150 155 160 Ser Val Leu Thr Phe Lys Val Leu Asp Leu Lys Asn Tyr Ile Asp Lys 165 170 175 Gln Leu Leu Pro Ile Leu Asn Lys Gln Ser Cys Ser Ile Ser Asn Ile 180 185 190 Glu Thr Val Ile Glu Phe Gln Gln Lys Asn Asn Arg Leu Leu Glu Ile 195 200 205 Thr Arg Glu Phe Ser Val Asn Ala Gly Val Thr Thr Pro Val Ser Thr 210 215 220 Tyr Met Leu Thr Asn Ser Glu Leu Leu Ser Leu Ile Asn Asp Met Pro 225 230 235 240 Ile Thr Asn Asp Gln Lys Lys Leu Met Ser Asn Asn Val Gln Ile Val 245 250 255 Arg Gln Gln Ser Tyr Ser Ile Met Cys Ile Ile Lys Glu Glu Val Leu 260 265 270 Ala Tyr Val Val Gln Leu Pro Leu Tyr Gly Val Ile Asp Thr Pro Cys 275 280 285 Trp Lys Leu His Thr Ser Pro Leu Cys Thr Thr Asn Thr Lys Glu Gly 290 295 300 Ser Asn Ile Cys Leu Thr Arg Thr Asp Arg Gly Trp Tyr Cys Asp Asn 305 310 315 320 Ala Gly Ser Val Ser Phe Phe Pro Gln Ala Glu Thr Cys Lys Val Gln 325 330 335 Ser Asn Arg Val Phe Cys Asp Thr Met Asn Ser Leu Thr Leu Pro Ser 340 345 350 Glu Val Asn Leu Cys Asn Val Asp Ile Phe Asn Pro Lys Tyr Asp Cys 355 360 365 Lys Ile Met Thr Ser Lys Thr Asp Val Ser Ser Ser Val Ile Thr Ser 370 375 380 Leu Gly Ala Ile Val Ser Cys Tyr Gly Lys Thr Lys Cys Thr Ala Ser 385 390 395 400 Asn Lys Asn Arg Gly Ile Ile Lys Thr Phe Ser Asn Gly Cys Asp Tyr 405 410 415 Val Ser Asn Lys Gly Val Asp Thr Val Ser Val Gly Asn Thr Leu Tyr 420 425 430 Tyr Val Asn Lys Gln Glu Gly Lys Ser Leu Tyr Val Lys Gly Glu Pro 435 440 445 Ile Ile Asn Phe Tyr Asp Pro Leu Val Phe Pro Ser Asp Glu Phe Asp 450 455 460 Ala Ser Ile Ser Gln Val Asn Glu Lys Ile Asn Gln Ser Leu Ala Phe 465 470 475 480 Ile Arg Lys Ser Asp Glu Leu Leu Ser Ala Ile Gly Gly Tyr Ile Pro 485 490 495 Glu Ala Pro Arg Asp Gly Gln Ala Tyr Val Arg Lys Asp Gly Glu Trp 500 505 510 Val Leu Leu Ser Thr Phe Leu 515 <210> 59 <211> 2088 <212> DNA <213> Artificial Sequence <220> <223> gH without the signal peptide <220> <221> misc_feature <222> (1077)..(1077) <223> C>A mutation at position 1077 <220> <221> misc_feature <222> (1)..(2082) <223> Ectodomain <220> <221> misc_feature <222> (2083)..(2088) <223> Transmembrane domain (truncated) <400> 59 cgatatggcg cagaagccgt atccgaaccg ctggacaaag cgtttcacct actgctcaac 60 acctacggga gacccatccg cttcctgcgt gaaaatacca cccagtgtac ctacaacagc 120 agcctccgta acagcacggt cgtcagggaa aacgccatca gtttcaactt tttccaaagc 180 tataatcaat actatgtatt ccatatgcct cgatgtcttt ttgcgggtcc tctggcggag 240 cagtttctga accaggtaga tctgaccgaa accctggaaa gataccaaca gagacttaac 300 acttacgcgc tggtatccaa agacctggcc agctaccgat ctttttcgca gcagctaaag 360 gcacaagaca gcctaggtga acagcccacc actgtgccac cgcccattga cctgtcaata 420 cctcacgttt ggatgccacc gcaaaccact ccacacggct ggacagaatc acataccacc 480 tcaggactac accgaccaca ctttaaccag acctgtatcc tctttgatgg acacgatcta 540 ctattcagca ccgtcacacc ttgtttgcac caaggctttt acctcatcga cgaactacgt 600 tacgttaaaa taacactgac cgaggacttc ttcgtagtta cggtgtccat agacgacgac 660 acacccatgc tgcttatctt cggccatctt ccacgcgtac ttttcaaagc gccctatcaa 720 cgcgacaact ttatactacg acaaactgaa aaacacgagc tcctggtgct agttaagaaa 780 gatcaactga accgtcactc ttatctcaaa gacccggact ttcttgacgc cgcacttgac 840 ttcaactacc tagacctcag cgcactacta cgtaacagct ttcaccgtta cgccgtggat 900 gtactcaaga gcggtcgatg tcagatgctg gaccgccgca cggtagaaat ggccttcgcc 960 tacgcattag cactgttcgc agcagcccga caagaagagg ccggcgccca agtctccgtc 1020 ccacgggccc tagaccgcca ggccgcactc ttacaaatac aagaatttat gatcacatgc 1080 ctctcacaaa caccaccacg caccacgttg ctgctgtatc ccacggccgt ggacctggcc 1140 aaacgagccc tttggacacc gaatcagatc accgacatca ccagcctcgt acgcctggtc 1200 tacatactct ctaaacagaa tcagcaacat ctcatccccc aatgggcact acgacagatc 1260 gccgactttg ccctaaaact acacaaaacg cacctggcct cttttctttc agccttcgca 1320 cgccaagaac tctacctcat gggcagcctc gtccactcca tgctggtaca tacgacggag 1380 agacgcgaaa tcttcatcgt agaaacgggc ctctgttcat tggccgagct atcacacttt 1440 acgcagttgt tagctcatcc acaccacgaa tacctcagcg acctgtacac accctgttcc 1500 agtagcgggc gacgcgatca ctcgctcgaa cgcctcacgc gtctcttccc cgatgccacc 1560 gtccccgcta ccgttcccgc cgccctctcc atcctatcta ccatgcaacc aagcacgctg 1620 gaaaccttcc ccgacctgtt ttgcttgccg ctcggcgaat ccttctccgc gctgaccgtc 1680 tccgaacacg tcagttatat cgtaacaaac cagtacctga tcaaaggtat ctcctaccct 1740 gtctccacca ccgtcgtagg ccagagcctc atcatcaccc agacggacag tcaaactaaa 1800 tgcgaactga cgcgcaacat gcataccaca cacagcatca cagtggcgct caacatttcg 1860 ctagaaaact gcgccttttg ccaaagcgcc ctgctagaat acgacgacac gcaaggcgtc 1920 atcaacatca tgtacatgca cgactcggac gacgtccttt tcgccctgga tccctacaac 1980 gaagtggtgg tctcatctcc gcgaactcac tacctcatgc ttttgaaaaa cggtacggta 2040 ctagaagtaa ctgacgtcgt cgtggacgcc accgacagtc gtctcctc 2088 <210> 60 <211> 696 <212> PRT <213> Artificial Sequence <220> <223> gH without the signal peptide <220> <221> MISC_FEATURE <222> (1)..(694) <223> Ectodomain <220> <221> MISC_FEATURE <222> (694)..(696) <223> Transmembrane domain (truncated) <400> 60 Arg Tyr Gly Ala Glu Ala Val Ser Glu Pro Leu Asp Lys Ala Phe His 1 5 10 15 Leu Leu Leu Asn Thr Tyr Gly Arg Pro Ile Arg Phe Leu Arg Glu Asn 20 25 30 Thr Thr Gln Cys Thr Tyr Asn Ser Ser Leu Arg Asn Ser Thr Val Val 35 40 45 Arg Glu Asn Ala Ile Ser Phe Asn Phe Phe Gln Ser Tyr Asn Gln Tyr 50 55 60 Tyr Val Phe His Met Pro Arg Cys Leu Phe Ala Gly Pro Leu Ala Glu 65 70 75 80 Gln Phe Leu Asn Gln Val Asp Leu Thr Glu Thr Leu Glu Arg Tyr Gln 85 90 95 Gln Arg Leu Asn Thr Tyr Ala Leu Val Ser Lys Asp Leu Ala Ser Tyr 100 105 110 Arg Ser Phe Ser Gln Gln Leu Lys Ala Gln Asp Ser Leu Gly Glu Gln 115 120 125 Pro Thr Thr Val Pro Pro Pro Ile Asp Leu Ser Ile Pro His Val Trp 130 135 140 Met Pro Pro Gln Thr Thr Pro His Gly Trp Thr Glu Ser His Thr Thr 145 150 155 160 Ser Gly Leu His Arg Pro His Phe Asn Gln Thr Cys Ile Leu Phe Asp 165 170 175 Gly His Asp Leu Leu Phe Ser Thr Val Thr Pro Cys Leu His Gln Gly 180 185 190 Phe Tyr Leu Ile Asp Glu Leu Arg Tyr Val Lys Ile Thr Leu Thr Glu 195 200 205 Asp Phe Phe Val Val Thr Val Ser Ile Asp Asp Asp Thr Pro Met Leu 210 215 220 Leu Ile Phe Gly His Leu Pro Arg Val Leu Phe Lys Ala Pro Tyr Gln 225 230 235 240 Arg Asp Asn Phe Ile Leu Arg Gln Thr Glu Lys His Glu Leu Leu Val 245 250 255 Leu Val Lys Lys Asp Gln Leu Asn Arg His Ser Tyr Leu Lys Asp Pro 260 265 270 Asp Phe Leu Asp Ala Ala Leu Asp Phe Asn Tyr Leu Asp Leu Ser Ala 275 280 285 Leu Leu Arg Asn Ser Phe His Arg Tyr Ala Val Asp Val Leu Lys Ser 290 295 300 Gly Arg Cys Gln Met Leu Asp Arg Arg Thr Val Glu Met Ala Phe Ala 305 310 315 320 Tyr Ala Leu Ala Leu Phe Ala Ala Ala Arg Gln Glu Glu Ala Gly Ala 325 330 335 Gln Val Ser Val Pro Arg Ala Leu Asp Arg Gln Ala Ala Leu Leu Gln 340 345 350 Ile Gln Glu Phe Met Ile Thr Cys Leu Ser Gln Thr Pro Pro Arg Thr 355 360 365 Thr Leu Leu Leu Tyr Pro Thr Ala Val Asp Leu Ala Lys Arg Ala Leu 370 375 380 Trp Thr Pro Asn Gln Ile Thr Asp Ile Thr Ser Leu Val Arg Leu Val 385 390 395 400 Tyr Ile Leu Ser Lys Gln Asn Gln Gln His Leu Ile Pro Gln Trp Ala 405 410 415 Leu Arg Gln Ile Ala Asp Phe Ala Leu Lys Leu His Lys Thr His Leu 420 425 430 Ala Ser Phe Leu Ser Ala Phe Ala Arg Gln Glu Leu Tyr Leu Met Gly 435 440 445 Ser Leu Val His Ser Met Leu Val His Thr Thr Glu Arg Arg Glu Ile 450 455 460 Phe Ile Val Glu Thr Gly Leu Cys Ser Leu Ala Glu Leu Ser His Phe 465 470 475 480 Thr Gln Leu Leu Ala His Pro His His Glu Tyr Leu Ser Asp Leu Tyr 485 490 495 Thr Pro Cys Ser Ser Ser Gly Arg Arg Asp His Ser Leu Glu Arg Leu 500 505 510 Thr Arg Leu Phe Pro Asp Ala Thr Val Pro Ala Thr Val Pro Ala Ala 515 520 525 Leu Ser Ile Leu Ser Thr Met Gln Pro Ser Thr Leu Glu Thr Phe Pro 530 535 540 Asp Leu Phe Cys Leu Pro Leu Gly Glu Ser Phe Ser Ala Leu Thr Val 545 550 555 560 Ser Glu His Val Ser Tyr Ile Val Thr Asn Gln Tyr Leu Ile Lys Gly 565 570 575 Ile Ser Tyr Pro Val Ser Thr Thr Val Val Gly Gln Ser Leu Ile Ile 580 585 590 Thr Gln Thr Asp Ser Gln Thr Lys Cys Glu Leu Thr Arg Asn Met His 595 600 605 Thr Thr His Ser Ile Thr Val Ala Leu Asn Ile Ser Leu Glu Asn Cys 610 615 620 Ala Phe Cys Gln Ser Ala Leu Leu Glu Tyr Asp Asp Thr Gln Gly Val 625 630 635 640 Ile Asn Ile Met Tyr Met His Asp Ser Asp Asp Val Leu Phe Ala Leu 645 650 655 Asp Pro Tyr Asn Glu Val Val Val Ser Ser Pro Arg Thr His Tyr Leu 660 665 670 Met Leu Leu Lys Asn Gly Thr Val Leu Glu Val Thr Asp Val Val Val 675 680 685 Asp Ala Thr Asp Ser Arg Leu Leu 690 695

Claims (38)

A composition comprising particles exhibiting an antigenic component, the composition comprising:
i) an antigenic component comprising a first peptide tag, and
ii) a moiety comprising a second peptide tag,
The composition wherein the antigenic component and moiety are linked through an isopeptide bond between the first and second peptide tags, and the antigenic component is greater than 50 kDa. The method of claim 1, wherein the antigenic component is 60 kDa, 70 kDa, 80 kDa, 90 kDa, 100 kDa, 110 kDa, 120 kDa, 130 kDa, 140 kDa, 150 kDa, 160 kDa, 170 kDa, 180 kDa, 190 A composition that is greater than kDa, 200 kDa, 300k Da or 400 kDa. The method according to claim 1 or 2, wherein the antigenic component is a monomer or a multimer, and optionally the multimer is a trimer, tetramer, pentamer, hexamer, sevenmer, octamer, ninmer or tenmer. Composition. The method of any one of claims 1 to 3, wherein the moiety is a virus, bacterium, multimerization scaffold for vaccination, multimerization to be a virus-like particle (VLP) A composition that is a protein component forming a, viral structural protein, a multimerization domain forming a nanoparticle, a synthetic nanoparticle or a synthetic VLP. The composition of any one of claims 1 to 4, wherein the moiety is HBsAg. The method of any one of claims 1 to 5, wherein the first and second peptide tags comprise a SpyTag and SpyCatcher pair; SnoopTag or SnoopTagJr and SnoopCatcher pair; RrgATag, RrgATag2 or DogTag and RrgACatcher pair, IsopepTag Pilin-C pair; IsopepTag-N and Pilin-N pair; PsCsTag and PsCsCatcher pair; And SnoopTagJr and DogTag pairs mediated by SnoopLigase; Or a variant, derivative or modification thereof, and optionally the first and second peptide tags are SpyTag/SpyCatcher pairs. The composition of any one of claims 1 to 6, wherein the antigenic component comprises an immunogenic component of HCMV pentameric. 8. The composition of claim 7, wherein the immunogenic component of the HCMV pentamer comprises one or more of gH, gL, pUL128, pUL130 or pUL131 subunits. The composition of claim 8, wherein the immunogenic component of the HCMV pentamer comprises all of the gH, gL, pUL128, pUL130 and pUL131 subunits. 10. The composition of any of claims 7-9, wherein the immunogenic component of the HCMV pentamer comprises a gH subunit having a truncated transmembrane domain. The method according to any one of claims 8 to 10, wherein the gH, gL, pUL128, pUL130 and pUL131 subunits have the amino acid sequences set forth in SEQ ID NOs: 28, 31, 35, 33 and 36, respectively, or functional equivalents thereof. Phosphorus composition. The composition according to any one of claims 8 to 11, wherein the first peptide tag is attached to the gH subunit, preferably at the C-terminus of the gH subunit. The composition of any one of claims 1 to 6, wherein the antigenic component comprises an immunogenic component of the RSV-F protein. The composition of claim 13, wherein the immunogenic component of the RSV-F protein is a pre-fusion F protein, optionally a stabilized pre-fusion F protein. The composition of claim 14, wherein the immunogenic component of the RSV pre-fusion F protein comprises a trimer of F ₁ and F ₂ subunits. The composition of any one of claims 13 to 15, wherein the RSV-F protein has the sequence set forth in any one of SEQ ID NOs: 50 to 58. 17. The composition of any one of claims 13 to 16, wherein the first peptide tag is attached to the C-terminus of the pre-fusion F protein. The composition of any of claims 1 to 17, wherein the first peptide tag is SpyTag. The composition of claim 18, wherein the SpyTag has the amino acid sequence set forth in SEQ ID NO: 30. The composition of claim 18 or 19, wherein the SpyTag is attached through a linker. The composition of claim 20, wherein the linker has the amino acid sequence set forth in SEQ ID NO: 29. 22. The composition of any of claims 1-21, wherein the moiety is HBsAg and optionally the second peptide tag is SpyCatcher. The composition of claim 22, wherein the second peptide is SpyCatcher and the SpyCatcher has the amino acid sequence set forth in SEQ ID NO: 38. The composition of claim 22 or 23, wherein the moiety is attached to the SpyCatcher via a linker, preferably a flexible linker. The composition of claim 24, wherein the linker has the amino acid sequence set forth in SEQ ID NO: 39. The composition of any one of claims 1 to 25, wherein the composition is an immunogenic or vaccine composition. A vaccine comprising a composition according to any one of claims 1 to 26 for use in the prevention and/or treatment of diseases. A method of preparing a composition according to any one of claims 1 to 26, comprising the following steps:
-Introducing into a first host cell a first nucleic acid encoding a first gene fusion of a first protein to a first peptide tag;
-Incubating said first host cell under conditions expressing said first gene fusion;
-Introducing a second nucleic acid encoding a second gene fusion of a second protein to a second peptide tag into a second host cell;
-Incubating the second host cell under conditions expressing the second gene fusion;
-Optionally purifying the expressed components;
-Incubating the expressed components under conditions that form an isopeptide bond between the first and second peptide tags; And optionally purifying the obtained composition. 29. The method of claim 28, wherein the first protein comprises an antigenic component and the second protein comprises a moiety. A nucleic acid molecule for use in the method according to claim 28 or 29, wherein the nucleic acid molecule encodes an amino acid sequence set forth in any one of SEQ ID NOs: 27 to 41. The nucleic acid molecule of claim 30 having the nucleotide sequence set forth in any one of SEQ ID NOs: 12 to 26 or 42 to 46. A nucleic acid molecule for use in the method according to claim 28 or 29, wherein the nucleic acid molecule encodes an amino acid sequence set forth in any one of SEQ ID NOs: 50 to 58. The nucleic acid molecule of claim 32 having the nucleotide sequence set forth in any one of SEQ ID NOs: 47-55. A vector comprising the nucleic acid molecule according to any one of claims 30 to 33. A host cell comprising a nucleic acid molecule according to any one of claims 30 to 32, or a vector according to claim 34. A kit comprising a composition comprising a first immunogenic composition and optionally one or more booster composition(s) comprising a second immunogenic composition, wherein the first and/or second immunogenic composition comprises claim 1 A kit comprising the composition according to any one of claims 26 to. A vaccine for use in the prevention and/or treatment of HCMV infection, comprising a composition according to any one of claims 7 to 12. A vaccine for use in the prevention and/or treatment of RSV infection, comprising a composition according to any one of claims 13 to 17.

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